Biomarker detection using integrated purification-detection devices

ABSTRACT

Techniques regarding integrated purification-detection devices for detecting one or more biomarkers are provided. For example, one or more embodiments described herein are directed to an apparatus, comprising a housing and a microfluidic chip contained within the housing. The microfluidic chip comprises a separation unit that separates, using one or more nano deterministic lateral displacement (nanoDLD) arrays, target biological entities having a defined size range from other biological entities included in a biological fluid sample. The microfluidic chip further comprises a detection unit that facilitates detecting presence of one or more biomarkers associated with the target biological entities using one or more detection molecules or macromolecules that chemically reacts with the one or more biomarkers.

BACKGROUND

The subject disclosure relates to integrated purification-detectiondevices for detecting one or more biomarkers, and more specifically, tointegrating lateral deterministic displacement arrays for particlepurification and one or more sensor arrays for biomarker detection ontoa single microfluidic chip.

Technologies capable of detecting the presence of biomarkers areubiquitous in biochemistry and a necessary element of diagnostic devicesin healthcare. Common methods of detection, such as enzyme-linkedimmunosorbent assays (ELISAs), utilize high-affinity interactionsbetween antibodies and their target epitope to achieve chemicalspecificity in detecting a particular analyte. One exemplary applicationof this method of revealing chemical specificity is targeting theepitopes of exosomes. Exosomes are extracellular vesicles (EVs) rangingin size from 30-150 nanometers (nm) found in minimally invasive andcompletely non-invasive biological fluids, or liquid biopsies, such asblood, urine, saliva, etc. Exosomes have emerged as a promising class ofbiomarkers for studying and identifying various disease conditions.These EVs contain a rich set of genetic information, includingtumor-specific proteins, micro ribonucleic acid (microRNA), messengerRNA (mRNA), and deoxy ribonucleic acid (DNA), that can individuallyand/or collectively provide a glimpse into the health state of anindividual at the sub-cellular level. To extract meaningful informationfrom these nanoscale prognosticators first requires the ability toisolate them from a complex biological fluid. Once they are extracted,some form of biochemical analysis or genetic sequencing is needed todetect presence of biomarkers. Both the extraction and detectionprocesses at present are cumbersome, costly, and impractical forfrequently running a diagnosis to catch a disease at an early stage orfor monitoring a patient's response to a particular treatment.

Focusing on the first requirement, the extraction piece, many standardbiochemistry methods have been applied to isolate exosomes, each withits own set of drawbacks, and, in general, the community is activelyseeking for better solutions to the sample preparation problem of EVs.The most common methods currently employed for the task includeultracentrifugation (UC), filtration, precipitation,immunoaffinity-based capture, nano deterministic lateral displacement(nanoDLD), Exodisc, viscoelastic flows, and exoTIC.

Ultracentrifugation (UC) exploits size differences between cells, EVs,and proteins to isolate these materials from each other usingprogressively higher spin speeds with intermediate extraction protocol.Major drawbacks are high spin speeds that can impact EV quality and longrun times (around 5 hours). UC is also a manual, batch process oftenresulting in lower exosome recovery and less than optimal EV quality.Filtration isolation techniques employ membrane filters, such aspolyvinylidene difluoride (PVDF) or polycarbonate filters, to sievecells and large EVs from biological samples. Filtration is sometimescoupled with ultracentrifugation to further separate exosomes fromproteins. These types of multistep arrangements require a bulkycentrifuge or vacuum system, use large sample volumes (30-100milliliters (mL)), require batch processing, and typically result inpoor yields due to clogging.

Several precipitation kit-based solutions have emerged to circumvent theneed for UC, including EXOEASY®, EXO-SPIN®, EXOQUICK® exosomeprecipitation, TOTAL EXOSOME ISOLATION REAGENT®, and/or PUREEXO®, toname a few. These products use special reagents to induce precipitationof exosomes, such as polyethylene glycol (PEG) based additives. Thesekits typically suffer from unacceptable purity due to polymercontamination, making downstream analysis difficult. These precipitationkits are also often limited to small, batched sample volumes.

The immunoaffinity-based capture isolation method specifically targetsexosomes from a complex biological fluid using, for example, tetraspaninproteins such as CD81 found on the surface of exosomes or markersspecific to the exosome's cell of origin to isolate them. A commontechnique utilizes antibody coated magnetic beads to capture exosomesthat contain specific markers from bodily fluids. These methods areexpensive, relying on specific antibodies that can vary batch to batchand suffer from stability issues. Thus, while these methods allowspecific subpopulations of exosomes to be isolated, the cost ofantibodies makes them generally unsuitable for isolating exosomes fromlarge quantities of biological samples.

In light of the inherent drawbacks surrounding the above-mentionedisolation standards, exploration of new solutions that can provide aroute toward a simple, inexpensive, automated, and rapid EV isolationtechniques have been reported in literature, including for example,lab-on-a-chip based approaches. Exemplary techniques within this realminclude nanoDLD, Exodisc, viscoelastic flows, and ExoTIC. NanoDLD refersto a technique wherein deterministic lateral displacement (DLD)technology is shrunk to the nanoscale, demonstrating the ability tosubfractionate exosome populations with tens of nanometers resolution ina continuous flow system (no batch processing) with a theory ofoperation. However, current nanoDLD techniques can only process very lowsample volumes at low throughput rates (e.g., about 0.2 microliters(μLs) per hour (hr)). Exodisc is a lab-on-a-disc separation techniquepresented in H.-K. Woo, et al., ACS Nano, vol. 11, pp. 1360, 2017. TheExodisc technique integrates two on-disc nanofilters that allow fullyautomated and label-free enrichment of EVs in the size range of 20-600nanometers (nm) within 30 minutes using a tabletop-sized centrifugalmicrofluidic system. Although the Exodisc technique have reportedlydemonstrated high yields (e.g., greater than 95% recovery of EVs fromcell culture and greater than a 100-fold higher concentration of mRNA ascompared with UC), the discs employed are large and costly. In addition,sample processing is batched rather than continuous flow, andsubfractionation of exosomes is not demonstrated or straightforwardlyapplicable.

Viscoelastic flow techniques have been used to isolate exosomes fromcell culture media and serum in a continuous flow, field-free, andlabel-free manner using an additive polymer (poly-oxyethylene or PEO) tocontrol the viscoelastic forces exerted on nanoscale EVs. As reported inC. Liu, et al., ACS Nano, vol. 11, pp. 6968, 2017, viscoelastic flowtechniques have demonstrated a separation purity greater than 90% with arecovery of greater than 80% and a throughput of 200 μ/hr. However,these techniques also suffer from disadvantages. In particular,viscoelastic flow devices are large (and thus more cumbersome andcostly), requiring channels of 32 millimeters (mm) in length to achievelateral resolution of particle streams (plus space for input/outports),and although isolation of 100 nm and 500 nm particles sizes have beenshown, this size selectivity does not lend itself to exosomefractionation.

The exosome total isolation chip (ExoTIC) filtration technique isanother exosome isolation technique reported in F. Liu, et al., ACSNano, vol. 11, pp. 10712-10723, 2017. ExoTIC employs a filtrationarrangement to achieve EV yields from 4 to 1000 fold higher than UCusing a low protein binding filter membrane from track-etchedpolycarbonate and a syringe pump driver at flowrates up to 30 mL/hrshown on 6 parallel syringes. A buffer wash step allows for EVpurification from smaller contaminates. Subfractionation is alsodemonstrated by staging filters down to the nanoscale and exosomes fromspecific cell lines are analyzed in terms of their size distribution.However, since filtration and purification are inherently sequentialprocesses, Exotic is a batch process requiring over 2 hours to perform asample preparation. In addition, nanoparticle tracking analysis (NTA)performed on ExoTIC subfractionated EV populations does not indicatestrong control of fractionated sizes, which calls into questionrun-to-run reliability.

Exosome detection and molecular profiling of exosomes presents an addedchallenge for exosome-based cancer diagnostics. Few technologies havearisen that attempt to tackle this problem. One technique described inH. Im, et al., Nat. Biotechnol., vol. 32, pp. 490-495, 2014, includes anano-plasmonic exosome (nPLEX) assay, which uses transmission surfaceplasmon resonance through periodic nanohole arrays functionalized withantibodies to profile the surface proteins of exosomes as well asproteins present in exosome lysates. This technique was successful atidentifying exosomes derived from ovarian cancer cells by theirexpression of CD24 and EpCAM with 100 times the sensitivity of an ELISA,and the exosomal and cellular protein profiles showed excellentcorrelation. However, upfront sample preparation was required for thenPLEX device to obtain a clean signal using standard UC or filtration.Another technique has been developed that uses an integratedmagneto-electrochemical sensor for exosome (iMEX) analysis. (See S.Jeong, et al., ACS Nano, vol. 10, pp. 1802-1809, 2016). The iMEXtechnique involves enriching exosomes directly from blood and profilingthem for molecular information. The platform uses magnetic selection andelectrochemical enrichment to isolate cell-specific exosomes fromcomplex media and achieved high sensitivity through magnetic enrichmentand enzymatic amplification to detect these markers electrically. Thistechnique however requires magnetic beads bearing horseradish peroxidase(HRP) labels to isolate the exosomes and produce a signal. In addition,off-platform sample preparation is required for each biomarker alongwith manual loading of the prepared sample onto each electrode.

SUMMARY

The following presents a summary to provide a basic understanding of oneor more embodiments of the invention. This summary is not intended toidentify key or critical elements or delineate any scope of theparticular embodiments or any scope of the claims. Its sole purpose isto present concepts in a simplified form as a prelude to the moredetailed description that is presented later. In one or more embodimentsdescribed herein, systems, apparatuses, and/or methods are provided thatrelate to integrated purification-detection devices for detecting one ormore biomarkers.

In accordance with various embodiments, an apparatus is provided thatcomprises a housing and a microfluidic chip contained within thehousing. The microfluidic chip comprises a separation unit thatseparates, using one or more nanoDLD arrays, target biological entitieshaving a defined size range from other biological entities included in abiological fluid sample. The microfluidic chip further comprises adetection unit that facilitates detecting presence of one or morebiomarkers associated with the target biological entities using one ormore detection molecules or macromolecules that chemically reacts withthe one or more biomarkers. In some implementations, the biologicalentities comprise exosomes. The biological entities can also includeother biological molecules and macromolecules ranging in size from 10.0nm to 200 nm, viruses, DNA sequences, RNA sequences and the like. Insome implementations, the one or more detection molecules ormacromolecules comprise an antibody or aptamer that binds with a targetepitope of the one or more biomarkers.

In various implementations, the detection unit can comprise a sensingelement, wherein a surface of the sensing element is coated with the oneor more detection molecules or macromolecules. With theseimplementations, the detection molecules or macromolecules canchemically react with the one or more biomarkers by binding to the oneor more detection molecules or macromolecules. In some implementations,based on the binding, the one or more detection molecules ormacromolecules generate a visual signal, such as a florescent signal.The sensing element can also comprise a signal enhancing structureselected from a group consisting of a photonic grating structure, aphotonic pillar array structure, an optoelectrical structure, and aplasmonic structure. In one or more implementations, a portion of thehousing formed adjacent to the sensing element is transparent orpartially transparent and enables visual observation of the fluorescentsignal.

The microfluidic chip can further comprise at least one conduit from theseparation unit to the detection unit that facilitates passage of bufferfluid comprising the target biological entities, as separated from theother biological entities, from the separation unit to the detectionunit. At least one inlet can be included on the microfluidic chipthrough which the buffer fluid passes from the conduit to the surface ofthe sensing element. In some implementations, the detection unit furthercomprises a blocking element formed at an interface between the surfaceof the sensing element and the at least one inlet. The blocking elementcan inhibit reverse flow of one or more reacted or unreacted molecularcomplexes (e.g., antibody/exosome complexes) from the surface of thesensing element through the at least one inlet, wherein the one or morereacted molecular complexes are formed as a result of a chemicalreaction between the one or more detection molecules or macromoleculesand the one or more biomarkers (e.g., an epitope on the surface of theexosomes). The microfluidic chip can further comprise at least oneoutlet from which the buffer fluid and unreacted portions of the targetbiological entities that fail to chemically react with the one or moredetection molecules or macromolecules, are excreted from the detectionunit.

In some implementations, the microfluidic chip further comprises atleast one inlet via which solution comprising the one or more detectionmolecules or macromolecules are injected into the detection unit to coatthe surface of the sensing element. In addition, in order to facilitatesimultaneous detection of a plurality of biomarkers, the detection unitcan comprise two or more separate detection chambers, wherein respectivechambers of the two or more separate detection chambers comprisedifferent types of detection molecules or macromolecules of the one ormore detection molecules or macromolecules. In this regard, thedifferent types of detection molecules or macromolecules can chemicallyreact with different types of biomarkers.

In another embodiment, a method is provided that comprises isolatingtarget biological entities (e.g., exosomes) having a defined size rangefrom other biological entities included in a biological fluid sampleusing a separation unit comprising one or more nanoDLD arrays formed ona microfluidic chip, thereby resulting in isolated target biologicalentities. The method further includes driving flow of a buffer fluidcomprising the isolated target biological entities through a conduit ofthe microfluidic chip from the separation unit to a sensing elementformed on the microfluidic chip, and facilitating detection of presenceof one or more biomarkers associated with the isolated target biologicalentities based on whether a detectable signal is generated by thesensing element in response to the driving.

For example, the sensing element can comprise one or more detectionmolecules or macromolecules, and the detectable signal can comprise areaction signal that is indicative of a chemical interaction between theone or more detection molecules or macromolecules and the one or morebiomarkers. For instance, the chemical reaction can include a reactionselected from a group consisting of a covalent bonding reaction, anelectrostatic interaction, a hydrophobic interaction, anantibody-epitope interaction, an aptamer-epitope reaction, aprotein-protein interaction, a protein-small molecule interaction, apolymerization reaction, a complementarity reaction, a complementary DNAstrand hybridization interaction, and a complementary RNA strandhybridization interaction. In some implementations, prior to thedriving, the method can comprise. Functionalizing a surface of thesensing element with the one or more detection molecules ormacromolecules, wherein the functionalizing comprises injecting asolution comprising the one or more detection molecules ormacromolecules into a chamber enclosing the surface of sensing elementvia at least one injection inlet of the microfluidic chip.

In some implementations of the subject method, the detectable signalcomprises a visual signal. With these implementations, the method canfurther comprise determining whether the detectable signal is generatedusing a microscope positioned adjacent the sensing element. The methodcan also include capturing, by a device operatively coupled to aprocessor, image data of the sensing element in association with thedriving, and determining, by the device, whether the visual signal isgenerated based on the image data.

In another embodiment, an apparatus is provided comprising a housing anda microfluidic chip contained within the housing. The microfluidic chipcomprises a separation unit that separates, using one or more nanoDLDarrays, exosomes from other biological entities included in a biologicalfluid sample, resulting in isolated exomes. The microfluidic chipfurther comprises a detection unit that facilitates detecting presenceof different biomarkers located on or within with the exosomes usingdifferent detection entities that respectively chemically react with thedifferent biomarkers, wherein the different detection entities areselected from a group consisting of molecules and macromolecules, and atleast one channel from the separation unit to the detection unit thatfacilitates flow of a buffer solution comprising the isolated exomes tothe detection unit. In some implementations, the detection unitcomprises different chambers that respectively detect presence of adifferent type of biomarker of the different types of biomarkers, andwherein the different chambers are respectively coated with a differentdetection entity of the different detection entities.

In one or more additional embodiments, a system is provided comprising amicrofluidic chip contained within a housing, wherein the microfluidicchip comprises a separation unit that separates, using one or morenanoDLD arrays, target biological entities having a defined size rangefrom other biological entities included in a biological fluid sample,resulting in isolated target biological entities. The microfluidic chipfurther comprises a detection unit that facilitates detecting presenceof one or more biomarkers associated with the isolated target biologicalentities using one or more detection molecules or macromolecules thatchemically react with the one or more biomarkers, and at least onechannel from the separation unit to the detection unit that facilitatesflow of a buffer solution comprising the isolated target biologicalentities to the detection unit. The system further comprises an imagingdevice (e.g., a microscope, a camera, etc.) that captures image data inassociation with flow of the buffer solution to the detection unit andcontact of the buffer solution with the one or more detection moleculesor macromolecules. In some implementations, the system further comprisesa memory that stores computer executable components, and a processorthat executes the computer executable components stored in the memory.The computer executable components can comprise an analysis componentthat evaluates the image data to determine biomarker informationregarding the presence of the one or more biomarkers. The computerexecutable components can also comprise a diagnosis component thatdetermines diagnostic information regarding a medical condition of apatient from which the biological fluid is sampled from based on thebiomarker information.

In yet another embodiment, a method is provided that comprises isolatingtarget biological entities having a defined size range from otherbiological entities included in a biological fluid sample using aseparation unit comprising one or more nanoDLD arrays, thereby resultingin isolated target biological entities, wherein the separation unit isformed on a microfluidic chip contained within a housing. The methodfurther comprises, driving flow of a buffer fluid comprising theisolated target biological entities through a conduit of themicrofluidic chip from the separation unit to a sensing element formedon the microfluidic chip, wherein the sensing element generates a visualsignal in response to detection of presence of one or more definedbiomarkers associated with the target biological entities. The methodfurther comprises capturing image data of the detection unit inassociation with the driving.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate a diagram of an example, non-limitingseparation-purification apparatus that integrates on-chip particlepurification and biomarker detection functionality in accordance withone or more embodiments described herein.

FIG. 2A presents a three-dimensional (3D) view of an examplemicrofluidic chip that integrates on-chip particle purification andbiomarker detection functionality in accordance with one or moreembodiments described herein.

FIG. 2B presents an orthogonal, two-dimensional (2D), perspective viewof an example microfluidic chip that integrates on-chip particlepurification and biomarker detection functionality in accordance withone or more embodiments described herein.

FIGS. 2C-2D present a 3D perspective view of an example detection unitof a microfluidic chip that integrates on-chip particle purification andbiomarker detection functionality in accordance with one or moreembodiments described herein.

FIG. 3A presents a 3D view of an example bottom plate of housing thatcouples with a microfluidic chip to facilitate on-chip particlepurification and biomarker detection functionality in accordance withone or more embodiments described herein.

FIGS. 3B and 3C present orthogonal, top-down views of an examplereservoir region that couples with a microfluidic chip to facilitateon-chip particle purification and biomarker detection functionality inaccordance with one or more embodiments described herein.

FIG. 4 illustrates a cross-sectional view of an example detection unitregion of a non-limiting separation-purification apparatus thatintegrates on-chip particle purification and biomarker detectionfunctionality in accordance with one or more embodiments describedherein.

FIG. 5 illustrates an enlarged view of an example separation unit of anexample microfluidic chip that integrates on-chip particle purificationand biomarker detection functionality in accordance with one or moreembodiments described herein.

FIG. 6 illustrates another enlarged view of an example separation unitof an example microfluidic chip that integrates on-chip particlepurification and biomarker detection functionality in accordance withone or more embodiments described herein.

FIG. 7 illustrates an enlarged view of an example detection unit of anexample microfluidic chip that integrates on-chip particle purificationand biomarker detection functionality in accordance with one or moreembodiments described herein.

FIG. 8 illustrates an enlarged view of another example detection unit ofan example microfluidic chip that integrates a blocking element inaccordance with one or more embodiments described herein.

FIG. 9A illustrates an enlarged view of another example detection unitof an example microfluidic chip that integrates on-chip particlepurification and biomarker detection functionality in accordance withone or more embodiments described herein.

FIG. 9B presents an orthogonal, 2D, perspective view of another examplehousing that couples with a microfluidic chip to facilitate on-chipparticle purification and biomarker detection functionality inaccordance with one or more embodiments described herein.

FIGS. 9C-9D present a 3D, perspective view of another example detectionunit of a microfluidic chip that integrates on-chip particlepurification and biomarker detection functionality in accordance withone or more embodiments described herein.

FIG. 10 illustrates an enlarged view of another example detection unitof an example microfluidic chip that integrates on-chip particlepurification and biomarker detection functionality in accordance withone or more embodiments described herein.

FIG. 11 illustrates an example system that facilitates integratingreal-time particle purification and biomarker detection in accordancewith one or more embodiments described herein.

FIG. 12 illustrates an example computing device that facilitatesreal-time biomarker detection and analysis in accordance with one ormore embodiments described herein.

FIG. 13 illustrates a flow diagram of an example, non-limiting methodfor performing particle purification and biomarker detection using anintegrated microfluidic device in accordance with one or moreembodiments described herein.

FIG. 14 illustrates a flow diagram of an example, non-limiting methodfor functionalizing a sensing element of an integrated microfluidicdevice and thereafter, employing the integrated microfluidic device toisolate exosomes and detect presence of exosomal surface markers basedon reaction with the functionalized sensing element, in accordance withone or more embodiments described herein.

FIG. 15 illustrates a flow diagram of an example, non-limiting methodthat facilitates integrating real-time particle purification andbiomarker detection in accordance with one or more embodiments describedherein.

FIG. 16 illustrates a block diagram of an example, non-limitingoperating environment in which one or more embodiments described hereincan be facilitated.

DETAILED DESCRIPTION

The following detailed description is merely illustrative and is notintended to limit embodiments and/or application or uses of embodiments.Furthermore, there is no intention to be bound by any expressed orimplied information presented in the preceding Background or Summarysections, or in the Detailed Description section.

Various embodiments described herein are directed to microfluidic chipdevices and systems that integrate biomarker detection together withupstream isolation and purification of biological entities, all on asingle chip, providing a powerful self-contained, and portable solutionto biochemical identification of disease-related biomarkers. Theintegrated purification-detection devices can be tailored to isolate anddetect biomarkers associated with various types of biological particles,including exosomes, as well as viruses and other biological entities. Inone or more embodiments, the microfluidic chip comprises as sensingelement that provides for real-time detection of one or many biomarkerslocated downstream of a continuous flow isolation and purificationseparation element that is also located on the microfluid chip. Byintegrating an upstream separation element with the sensing element, thenoise floor of the sample is minimized to enhance sensitivity byremoving background contaminates and larger unwanted material, such ascellular debris and multi-vesicular bodies (MBVs).

The separation element can employ an arrangement of multiplexed lateraldeterministic displacement (DLD) arrays, (e.g., nanoDLD arrays) for abuffer exchange of target biomolecules (e.g., exosomes) from an inputsample with smaller contaminants, such as small molecules, proteins, andsalts, exiting a common set of waste outlets. The nanoDLD arrays can beconfigured to bump or otherwise direct purified target biologicalentities into the portion of the buffer medium that flows into a commonbus toward the downstream sensing element. In various embodiments, thesensing element can provide for detecting presence of one or morebiomarkers present on the surface of the purified target biomoleculesvia chemical specificity between the one or more biomarkers and anotherchemical coated on the surface of the sensing element. For example, thesensing element can be coated with antibodies having a chemicalspecificity for a known epitope that may be present on the surface ofisolated exosomes. In some implementations, the sensing element canincorporate a plurality of different antibodies that provide forsimultaneous detection of two or more biomarkers. Simultaneous detectionof multiple markers allows for fast, effective diagnosis of disease,such as certain forms of cancer. However, the sensing element biomarkerdetection methods may be more broadly extended to any specific chemicalor biochemical interaction between two molecules or macromolecules, canbe naturally occurring or synthetic and can be a permanent covalentlinkage or a reversible bond (e.g., electrostatic interactions,hydrophobic interactions, complementarity, etc.).

In various exemplary embodiments, the sensing element can be located ator near the center of the chip and provide for optical readout ofchemical reactions indicative of biomarker presence. For example, thepurified sample can flow from the common bus mover the sensing element,which can include a signal-enhancing element such as a photonic grating,an optoelectrical element or plasmonic structure, coated with antibodiesor aptamers known to bind with target epitopes or surface markers. Forinstance, the sensing element can be configured to detected chemicalreactions that produce a fluorescent signal that is observable withfluorescence microscopy, and therefore manually detectable by eye orthrough software to automate the process. Accordingly, the sensingelement can provide for real-time monitoring and diagnosis of aparticular disease condition.

In one or more embodiments, the disclosed microfluidic chip can becoupled to a housing to facilitate a real-time exosome separation andbiomarker detection process. For example, one exemplary process caninclude loading (e.g., pipetting) several fluids into various reservoirsonto the housing containing a microfluidic chip. The fluids include abiological sample (e.g., urine, blood, saliva, etc.), a buffer, and oneor more fluids containing antibody or aptamer chemistries for surfacefunctionalization of all or dedicated parts of the on-chip sensingelement. A pressure-driven can be used to first drives the antibody oraptamer containing fluids onto the sensing element to functionalize thesurface for immunocapture of exosomes containing certain target surfacemarkers. Next, the biological sample and buffer can be pressure drivenonto the chip where exosomes are harvested and purified using thenano-DLD arrays of the detection unit, and then captured on thedownstream sensing element for detection and analysis using fluorescencemicroscopy, either manually by an operator or using a software analysisprogram.

In this regard, the subject integrated purification-detection devicesand systems provide an all-in-one solution for sample preparation fromcomplex patient fluids together with detection of multiple surfacemarkers all on a single chip. The disclosed exosome isolation andbiomarker detection devices provide a uniquely powerful, self-contained,and portable solution to biochemical identification of disease-relatedexosomal cohorts for biomarker discovery and diagnostic applications.Thus, the technology provides a means of semi-automating the biomarkerdiscovery process as well as aids in rapid sample screening that canpotentially be performed at the clinic.

As used herein, the term lab-on-a-chip (LOC) can refer to one or moredevices that can integrate one or more laboratory functions onto anintegrated circuit (e.g., a semiconductor substrate structure) toachieve autonomous screening of one or more samples. LOCs can utilizemicroelectromechanical systems and/or microfluidic systems to facilitatescreening the one or more samples. One of ordinary skill in the art willrecognize that a LOC devices can range in size from, for example, one ormore square millimeters to one or more square centimeters. One or moreembodiments can utilize microfluidics in a LOC device to detect one ormore target biomarkers, wherein the biomarkers can be indicative ofvarious traits (e.g., physical properties) and/or health conditions(e.g., diseases). Thus, in some embodiments, the one or more integratedpurification-detection devices described herein can be considered LOCdevices that can facilitate biomarker detection, wherein the one or moreLOC devices can be operated quickly (e.g., near instantaneously), in avariety of locations (e.g., at an entity's home), and without thetypical need for specialized laboratory equipment.

As used herein the term deterministic lateral displacement (DLD) canrefer to one or more microfluidic techniques that can size fractionate apolydisperse suspension of molecules through the use of one or morearrays of obstacles. For example, DLD arrays can laterally displacetarget molecules within a sample stream based on size. Further, DLDarrays can comprise a plurality of pillars arranged in a latticestructure. Rows of pillars comprising the lattice structure can bepositioned offset of each other at a defined angle, and pillars can beseparated from each other by a defined gap size. The defined angleand/or gap size can facilitate displacement of one or more molecules ofa target size range comprised within a stream flowing through the DLDarray.

As used herein the term nanoDLD array can refer to a DLD array that canbe characterized by one or more dimensions ranging from greater than orequal to 1 nanometer (nm) and less than or equal to 999 nm. For example,a nanoDLD array can be a DLD array characterized by a gap size (e.g., adistance between adjacent pillars comprised within the latticestructure) of greater than or equal to 1 nm and less than or equal to999 nm (e.g., greater than or equal to 25 nm and less than or equal to235 nm). In one or more embodiments, a nanoDLD array can facilitatedisplacement of exosomes, viruses, and other biomolecules ormicromodules of various sizes (e.g., from 1 nm to 999 nm). In someimplementations, the nanoDLD arrays described herein can also isolategenetic code sequences that can be characterized as having an exemplarylength ranging from, but not limited to, greater than or equal to 25base pairs (bp) and less than or equal to 200 bp.

As used herein, unless otherwise specified, terms such as on, overlying,atop, on top, positioned on, or positioned atop mean that a firstelement is present on a second element, wherein intervening elements maybe present between the first element and the second element. As usedherein, unless otherwise specified, the term directly used in connectionwith the terms on, overlying, atop, on top, positioned, positioned atop,contacting, directly contacting, or the term direct contact, mean that afirst element and a second element are connected without any interveningelements, such as, for example, intermediary conducting, insulating orsemiconductor layers, present between the first element and the secondelement. As used herein, terms such as upper, lower, above, below,directly above, directly below, aligned with, adjacent to, right, left,vertical, horizontal, top, bottom, and derivatives thereof shall relateto the disclosed structures as oriented in the drawing figures.

One or more embodiments are now described with reference to thedrawings, wherein like referenced numerals are used to refer to likeelements throughout. In the following description, for purposes ofexplanation, numerous specific details are set forth in order to providea more thorough understanding of the one or more embodiments. It isevident, however, in various cases, that the one or more embodiments canbe practiced without these specific details. Further, it is to beunderstood that common cross-hatching and/or shading depicted across thedrawings can represent common features, compositions, and/or conditionsdescribed herein in accordance with one or more embodiments.

FIGS. 1A and 1B illustrates a diagram of an example, non-limiting,separation-purification apparatus 100 that integrates on-chip particlepurification and biomarker detection functionality in accordance withone or more embodiments described herein. As shown in FIG. 1A, theseparation-purification apparatus 100 comprises a microfluidic chip 106provided within a housing 102. The housing is composed of a top plate102A and a bottom plate 102B. FIG. 2B depicts theseparation-purification apparatus 100 with the top plate 102A removed.In various embodiments, the housing 102 can be or correspond to a flowcell or other form of packaging that houses the microfluidic chip 106.For example, in some embodiments, the top plate 102A and the bottomplate 102B can be physically coupled to one another (e.g., via one ormore screws or another suitable attachment mechanism) with themicrofluidic chip 106 sandwiched therebetween. In the embodiment shown,the top plate 102A is transparent or semitransparent. For example, thetop plate 102A can be formed of a clear acrylic plastic, glass, oranother suitable material. The bottom plate 102B can also be formed witha transparent or semitransparent material, such as clear acrylicplastic, glass or another suitable material. In other embodiments, thebottom plate 102B can be formed with a non-transparent material, such assilicon or another material in which microchannels, reservoirs, vias,etc., can be fabricated thereon and/or therein.

The microfluidic chip 106 comprises a substrate material with aplurality of elements formed on or within the substrate material thatfacilitate on-chip particle filtration and biomarker detection. Forexample, in some embodiments, the microfluidic chip 106 can comprise asilicon substrate with elements formed therein and/or thereon usingvarious semiconductor fabrication techniques. Other suitable materialsfor the microfluidic chip 106 can include glass, plastic, or acombination thereof. The elements formed on and/or within themicrofluidic chip 106 can include a separation unit that includes one ormore DLD arrays and/or nanoDLD configured to separate particles ofinterest (e.g., exosomes) from other particles included in a biologicalfluid sample. The biological fluid sample can include for example (butis not limited to), a blood sample, a urine sample, a tissue sample, asaliva sample, a plasma sample, a cell culture medium, an in vitrosample, a plant sample, a food samples, a combination thereof, and/orthe like. The microfluidic chip 106 further includes a detection unitthat facilitates detecting one or more biomarkers located on or withinthe particles of interest using a sensing element 108. For example, inone or more embodiments, the sensing element 108 can be coated with oneor more detection molecules or macromolecules configured to chemicallyreact with the one or more biomarkers. In accordance with thesesembodiments, the detection unit can facilitate flowing solutioncomprising the isolated particles of interest over the sensing element108. If the one or more biomarkers are present, the one or moredetection molecules or macromolecules will chemically react with thebiomarkers and produce some form of detectable signal (e.g., a visualsignal) that can be read from the sensing element 108. The microfluidicchip 106 further includes a microfluidic busing network consisting of aplurality of microchannels, busses, vias and/or reservoirs formed on orwithin the microfluidic chip. The microfluidic bussing networkfacilitates transporting fluid streams between the separation unit, thedetection unit, and other elements present on or within the microfluidicchip 106.

As shown in FIG. 1A, the top plate 102A can include a window region 124formed on or within the top surface of the top plate 102A. This windowregion 124 can comprise glass or another transparent material (e.g., inimplementations in which the material employed for the top plat 102 issemitransparent) that facilitates clearly visualizing the sensingelement 108 of the microfluidic chip 106. For example, in someimplementations, the window region 124 can be formed with transparentglass and the remainder of the top plate 102A can be formed withtransparent or semitransparent acrylic plastic. The window region can beformed in an area of the top plate that is aligned with the sensingelement 108 when the top plate 102A is attached to the bottom plate102B. In some embodiments, (as shown in FIG. 1A and more clearly shownin FIG. 1B), a capping layer 110 can be formed on the top surface of themicrofluidic chip 110. The capping layer 110 can comprise a transparentmaterial (e.g., glass, acrylic plastic, etc.) that provides forfluidically sealing the microfluidic elements formed on the top surfaceof the microfluidic chip 110.

FIG. 2A presents an example 3D view of microfluidic chip 106 asseparated from separation-purification apparatus housing in accordancewith one or more embodiments described herein. In the embodiment shown,the capping layer 110 is also removed. FIG. 2B presents an orthogonal,2D, perspective view of microfluidic chip 106 taken along axis B-B′shown in FIG. 2A, in accordance with one or more embodiments describedherein. Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

As shown in FIG. 2B, the microfluidic chip 106 can include a circulararchitecture with several elements formed around the sensing element 108provided at or near the center of the chip. In particular, (shown inlight grey), the microfluidic chip 106 can include an inlet bus 204formed around an outer perimeter area of the chip and fluidicallycoupled to a global inlet via 202. For example, the inlet bus 204 can beetched or otherwise formed within a portion of the thickness of thechip. In some implementations, the inlet bus 204 can be etched deeperthan other fluidic channels and/or elements formed within the thicknessof the chip. For example, in some implementations, the base of the inletbus 204 can be located 100 μm (or greater) from the bottom surface ofthe microfluidic chip (e.g., the surface opposite the capping layer110), without penetrating the bottom surface of the microfluidic chip.The global inlet via 202 can however penetrate through the bottomsurface of the microfluidic chip to facilitate receiving andtransporting fluid therethrough and into the inlet bus 204. For example,in various embodiments, the inlet bus 204 can be configured to receivebiological sample fluid introduced through the global inlet via 202, anddistribute the biological sample fluid evenly, (or substantially evenly)throughout the inlet bus 204 (e.g., in the direction shown via thedashed arrows extending from the global inlet via 202).

The microfluidic chip 106 further includes a separation unit 206 formedaround the sensing element 108 and within the perimeter of the inlet bus204. For example, in the embodiment shown, the separation unit 206 isdivided into four segments respectively arranged in ring shape (or moreaccurately, a rectangular shape) within the perimeter of the inlet bus.However, it should be appreciated that the specific shape or geometricalconfiguration of the separation unit 206 can vary. In the embodimentshown, the separation unit 206 encompasses the alternating black andwhite checkered lines formed parallel to one another, as well as thedark grey region formed in between them. As discussed in greater detail,the dark grey region of the separation unit 206 can comprise a pluralityof DLD or nanoDLD arrays configured to separate target particles of aparticular size range from other particles included in the biologicalsample fluid, and the respective checked lines can correspond to inletand outlet vias through which fluid passes into and out of the DLD ornanoDLD array.

For example, in some implementations, the first or outermost checkeredline provided at the interface between the inlet bus 204 and the DLD ornanoDLD region (e.g., the dark grey region), can include a plurality ofopenings through which the biological fluid sample can flow from theinlet bus 204 and into the DLD or nanoDLD array, (e.g., in the directionshown by the dashed arrows). In some implementations, the firstcheckered line can also include a plurality of second inlet vias throughwhich another fluid, such as a buffer fluid, can be introduced. Forexample, as discussed in greater detail infra, as the biological fluidand the buffer fluid can simultaneously flow through the DLD or nanoDLDarrays, the particles of interest can be bumped into or otherwisecaptured in first streams of the buffer fluid. Other undesired particlesincluded in the biological fluid sample can be captured in secondstreams of the biological fluid sample that generally flow in a straighttrajectory through the DLD or nanoDLD arrays. For example, inimplementations in which the target particles include exosomes, theundesired particles removed by the separation unit can includepotentially contaminating small molecules such as salts, proteins,lipids and the like. The second or innermost checked line (providedadjacent to outlet bus 208), can further include a plurality of openingsthrough which the first streams can exit the DLD or nanoDLD array (e.g.,the dark grey region of the separation unit 206) and enter into outletbus 208 (e.g., in the direction shown by the dashed arrows). The secondchecked line can also include a plurality of outlet vias through whichthe respective second streams can be collected and expelled from themicrofluidic chip 106 (e.g., as waste fluid).

The outlet bus 208 comprises an etched channel formed within thethickness of the microfluidic chip 106. The outlet bus 208 can receivethe filtered stream of the buffer fluid including the target particlesfrom the separation unit 206 and transport the filtered target particlestream to the downstream, sensing element 108 (e.g., in the directionshown by the dashed arrows. In one or more embodiments, the interface(or interfaces) between the outlet bus 208 and the sensing element 108can include one or more openings (not shown) through which the targetparticle buffer stream can enter and flow onto and over the sensingelement (e.g., in the direction shown by the dashed grey arrows). Thesensing element 108 can further include a global outlet via 210 throughwhich the buffer stream can exit the microfluidic chip 106, along withany unreacted and/or unbound particles included in the filtered, targetparticle buffer stream. In various embodiments, the outlet bus 208 andthe inlet bus 204 bus can be etched deeper than both the separation unit206 and the sensing element 108. The purpose for this is to ensure thatfluidic resistance is dropped or decreased across the separation unit206 and the sensing element 108.

The microfluidic chip 106 also include one or more third inlet vias 212that are fluidically coupled to the sensing element 108. In theembodiment shown, four third inlet vias 212 are shown, however thenumber of third inlet vias 212 can vary. The one or more third inletvias 212 can facilitate introducing a detection fluid onto the sensingelement 108 for coating and functionalizing the sensing element 108. Forexample, the detection fluid can include one or more types of detectionmolecules or macromolecules (e.g., antibodies, aptamers, etc.), known tochemically react with one or more biomarkers of interest that may bepresent on or within the separated particles of interest. In someimplementations, prior to injecting the biological sample fluid into themicrofluidic chip, the detection fluid can be injected through the oneor more third inlet vias 212 and flowed onto the sensing element 108 tocoat and functionalize the sensing element 108. Excess detection fluidor otherwise portions of the detection fluid that do not coat thesurface of the sensing element 108 can also flow through the globaloutlet via 210.

In the embodiment shown, a circular, distribution bus 214 can be formedaround the perimeter of the sensing element 108 (depicted by the thingrey line formed around the sensing element 108) to facilitate evenlydistributing the detection fluid and the biological fluid over thesurface of the sensing element 108. For example, the distribution bus214 can be formed around the perimeter of the sensing element 108 andminimize fluidic resistance to induce uniform fluid flow from theperimeter injection sites to the center of the sensing element 108,thereby enabling uniform coverage of coating chemistry and sample overthe sensing element during device operation.

In various embodiments, the sensing element 108, the portion of theoutlet bus 208 that connects to the sensing element 108, the globaloutlet via 210, the one or more third inlet vias 212, and thedistribution bus 214, can constitute the detection unit of the subjectmicrofluidic chips (e.g., microfluidic chip 106).

In this regard, FIGS. 2C and 2D present a 3D, perspective view of anexample detection unit 200 of a microfluidic chip (e.g., microfluidicchip 106) that integrates on-chip particle purification and biomarkerdetection functionality in accordance with one or more embodimentsdescribed herein. Repetitive description of like elements employed inrespective embodiments is omitted for sake of brevity.

As shown in FIG. 2C with respect to the dashed arrow lines, detectionfluid can be introduced at the respective third inlet vias 212 andflowed over the sensing element 108 and out the global outlet via 210 tocoat and/or functionalize the surface of the sensing element 108. Asshown in FIGS. 2C and 2D, the outlet bus 208 can comprise a plurality ofdeeply etched channels that connect to the sensing element 108. As shownin FIG. 2D with reference to the dashed arrow lines, after the sensingelement has been functionalized, a stream of buffer fluid comprisingpurified target particles can flow from the separation unit 206, upthrough the outlet bus 208 channels and onto the sensing element 108.Excess detection fluid and target particle buffer stream can furtherflow into the global outlet bus 210 to be removed from the microfluidicchip 106.

With reference again to FIGS. 1A and 1B in connection with reference toFIGS. 2A-2D, in various embodiments, the microfluidic bussing network(e.g., including the global inlet via 202, the inlet bus 204, the secondinlet vias (not shown) for introducing the buffer fluid into theseparation unit 206, the plurality of outlet vias (not shown) forremoving waste fluid from the separation unit 206, the outlet bus 208,the global outlet via 210, and/or the one or more third inlet vias 212)can be fluidically coupled to one or more fluid inlets and outletsprovided within the bottom plate 102B of the housing 102 via which themicrofluidic chip receives and excretes fluids. For example, in one ormore embodiments, the housing 102 can be or include a flow cell oranother form of packaging that facilitates flowing or otherwiseinjecting fluid into one or more input vias connected to the bussingnetwork of the microfluidic chip 106 and removing fluid from themicrofluidic chip 106. The housing 102 can be formed with variousmaterials, including silicon, glass, plastic, or a combination thereof.In this regard, the bottom plate 102B of the housing 102 can include oneor more inlet ports through which fluid is injected (e.g., using asyringe, pipette, or the like) into one or more flow cell channels (notshown) and/or reservoirs (not shown) provided on or within the bottomplate 102B. The one or more flow cell channels/and reservoirs can befluidically coupled to the microfluidic bussing network of themicrofluidic chip 106. The bottom plate 102B can further include one ormore output ports through which fluid is exported or otherwise removedfrom the microfluidic chip 106 and/or one or more reservoirs of thehousing 102. In this regard, one or more fluids can flow into the bottomplate 102B of separation-purification apparatus 100, through themicrofluidic chip 106, and then out of the microfluidic chip via thehousing 102.

For example, FIG. 3A presents a 3D view of the bottom plate 102B of thehousing 102 as separated from the microfluidic chip 106 and the topplate 102A. Repetitive description of like elements employed inrespective embodiments is omitted for sake of brevity.

With reference to FIG. 3A, in conjunction with reference to FIGS. 1A-1Band 2A-2D, in the embodiments shown, the bottom plate 102B of thehousing can include a chip pocket 312 that can receive the microfluidicchip 106. In this regard, the microfluidic chip 106 can be inserted intothe chip pocket 312 such that a bottom surface (e.g., the surfaceopposite the sensing element 108), is opposed to the upper surface ofthe bottom plate 102B. The bottom surface of the microfluidic chip 106can further include openings or vias which can correspond to one or moreinlet vias and outlet vias of the microfluidic chip (e.g., the globalinlet via 202, the global outlet via 210, and other vias describedbelow). In some embodiments, these openings or vias in/through thebottom surface of the microfluid chip can align with and fluidicallycouple to corresponding fluid inlets/outlets provided by the bottomplate 102B of the housing.

The bottom plate 102B includes three inlet ports or capillaries,including inlet port 112, inlet port 114 and inlet port 116. These inletports can respectively be used to inject fluid (e.g., the biologicalfluid sample, the buffer fluid, and the detection fluid), into themicrofluidic chip. The bottom plate 102B also includes two outlet ports,outlet port 118 and outlet port 120. These outlet ports can respectivelybe used to remove fluid (e.g., waste fluid, excesses detection fluid,and purified sample fluid as it flows over the sensing element 108 andout through the global outlet via 210), from the microfluidic chip 106and the bottom plate 102B. In some embodiments, a single outlet port canbe used. In other embodiments, more than two output ports can be used.In the embodiment shown, the inlet ports and outlet ports are depictedas tubes that extend from sides of the bottom plate 102B. However, itshould be appreciated that the location of the respective inlet andoutlet ports can vary. In addition, although the inlet ports are shownas tubes, it should be appreciated that these tubes connect tocorresponding openings/microfluidic channels (not shown) formed withinthe body of the bottom plate. In this regard, the tubes can be removablyattached/detached from corresponding openings/microfluidic channels inthe bottom plate 102B.

For example, in various embodiments, an upper surface region of thebottom plate 102B can include a plurality of fluidic connections andfluid reservoirs which can receive fluid from the one or more inletports (e.g., inlet port 112, inlet port 114 and/or inlet port 114) forintroducing into the microfluid chip, and/or receive fluid as it isexcreted from the microfluid chip. For example, in the embodiment shown,these fluidic connections/reservoirs respectively include fluidicconnection 302′, buffer fluid reservoir 304, waste fluid reservoir 306,detection fluid reservoir 308, and fluidic connection 310′. Therespective reservoirs, including the buffer fluid reservoir 304, thewaste fluid reservoir 306, and the detection fluid reservoir 308, can befluidic pools that can contain a fluid within. Specifically, in one ormore embodiments, the buffer fluid reservoir 304 can receive and containbuffer fluid for injection into the separation unit of the microfluidicchip, the waste fluid reservoir 306 can receive and contain waste fluid(comprising unwanted particles) removed by the separation unit, and thedetection fluid reservoir 308 can receive and contain detection fluidcomprising the surface chemistry molecules/macromolecules for coatingthe sensing element 108. Each of these reservoirs can include one ormore openings (not shown) through which the corresponding fluid can beinjected into the reservoir and one or more openings (not shown) throughwhich fluid can removed from the reservoir.

In the embodiment shown, the buffer fluid reservoir 304, waste fluidreservoir 306, detection fluid reservoir 308, are formed on/within anupper surface region of the bottom plate 102B. For example, in someimplementations, the respective reservoirs can be exposed on the topsurface of the bottom plate on the housing. With these embodiments, thereservoirs can become enclosed by the bottom surface of the microfluidicchip when the microfluidic chip is inserted into the chip pocket 312. Inthis regard, when the microfluidic chip is inserted into the chippocket, the bottom surface of the microfluidic chip can cover andenclose the reservoirs. In other implementations, a top surface of thebottom plate 102B can enclose the reservoirs. In another embodiment, oneor more of these reservoirs can be formed within the microchip 106and/or an intermediary layer (not shown) between the microchip 106 andthe bottom plate 102B. In various embodiments, these three reservoirsare collectively referred to herein as the reservoir region.

In one or more embodiments, fluidic connection 302′ can correspond to anopening in an upper surface of the bottom plate 102B that can align withand connect to the global inlet via 202 of the microfluid chip. Inaccordance with this example embodiment, the inlet port 112 can connectto the fluidic connection 302′ and the global inlet via 202 of themicrofluidic chip 106 when the microfluid chip 106 is inserted into thechip pocket 102. In this regard, inlet port 112 can be configured toreceive a biological fluid sample and facilitate flowing the biologicalfluid sample through a channel (not shown) formed within the bottomplate 102B that connects to the fluidic connection 302′ and which isfurther aligned with and connects to the global inlet via 202 of themicrofluidic chip 106. In one or more embodiments, the interface betweenthe fluidic connection 302′ and the global inlet via 202 can employ ano-ring seal or gasket to maintain fluidic isolation between thereservoirs (via compressive pressure applied to the o-ring seal orgasket) and controlling passage of the biological fluid from the bottomplate 102B, through global inlet via 202 and into the inlet bus 204. Theinlet bus 204 can further receive the biological fluid and pass thefluid through the microfluidic chip 106 for processing by the separationunit and the detection unit of the microfluidic chip 106, as herein. Insome implementations, the biological fluid sample can be injected intothe inlet port 112 via a pipette, via a syringe, or via from anotheroff-chip biological sample reservoir connected to the inlet port 112 viaa suitable tube or capillary. In various embodiments, the biologicalfluid sample can be injected through the inlet port 112 and into themicrofluidic chip 106 (e.g., via the first global inlet via) using apressure driving system or device (not shown) that is external to theseparation-purification apparatus 100.

Similarly, in some embodiments, the fluidic connection 310′ cancorrespond to an opening in an upper surface of the bottom plate 102Bthat can align with and connect to the global outlet via 210 of themicrofluid chip. In accordance with this example embodiment, the outletport 120, can be fluidically connected to the fluidic connection 310′via a microfluidic channel (not shown) formed within the bottom plate102B (and through the center of the detection fluid reservoir 308). Thefluidic connection 310′ can further be fluidically connected to theglobal outlet via 210 of the microfluidic chip 106 when inserted intothe chip pocket 312. In this regard, the outlet port 120 can beconfigured to export fluid passed over the sensing element 108 andflowed into the global outlet via 120. For example, in someimplementations, this fluid can initially include excess detection fluidthat flows from inlet port 116 through separation-purification apparatus100, over the sensing element 108 of the microfluidic chip 106 and exitsthe separation-purification apparatus 100 via outlet port 120. In thisregard, outlet port 120 can provide for removing excesses reagentchemistry (e.g., antibodies, aptamers, etc.) from the sensing element108 in association with the coating process used to functionalize thesensing element 108. Outlet port 120 can also be employed to remove thestream of buffer fluid including separated particles of interest as thestream is passed over the sensing element 108 to detect presence ofbiomarkers on or within the particles of interest. In this regard, astream of buffer fluid including separated particles of interest canflow from the separation unit 206 of the microfluidic chip 106 to thedownstream detection unit and over the sensing element 108 in a steadymanner, allowing for biomarkers to contact and react with the sensingelement 108, while unreacted or unbound particles in the buffer streamare excreted through the outlet port 120. In some implementations, exitof fluid through the global outlet via 210 can be contained via ano-ring or another suitable gasket formed around and//or within theglobal outlet via 210 and/or the fluidic connection 310′.

The introduction of buffer fluid and detection fluid into themicrofluidic chip 106 via the corresponding buffer fluid reservoir 304and detection fluid reservoir 308 (when the microfluidic chip isinserted into the chip pocket 312), and the removal of waste fluid fromthe microfluidic chip 106 via the corresponding waste fluid reservoir306, is discussed with reference to FIGS. 3B and 3C in connection withFIGS. 1A-1B, 2A-2D and 3A.

In this regard, FIGS. 3B and 3C present orthogonal, 2D, top-down viewsof an example reservoir region that couples with a microfluidic chip tofacilitate on-chip particle purification and biomarker detectionfunctionality in accordance with one or more embodiments describedherein. The reservoir region can comprise three reservoirs including thebuffer fluid reservoir 304, waste fluid reservoir 306, and detectionfluid reservoir 308. Each of the reservoirs can be enclosed fluidicpools that can contain a fluid within. In this regard, each of thesefluid reservoirs can be defined by an upper surface, a bottom surface,and a fluidic space between the bottom surface and the upper surface.FIG. 3B depicts the bottom surface 300 of the reservoir region. FIG. 3Cdepicts the upper surface 301 of the reservoir region. In the embodimentshown in FIG. 3A, the reservoir region is formed within an upper portionof the bottom plate 102B. However, the specific location of therespective reservoirs can vary so long as they are located between theactive features of the microfluidic chip (e.g., the separation unit andthe detection unit) and the corresponding fluidic inlets/outlets of thebottom plate 102B. In this regard, in some embodiments, the bottomsurface of the 300 of the reservoir region can be part defined withinthe bottom plate 102B and the upper surface 300 of the reservoir regioncan also be defined by/within the bottom plate (e.g., the upper surfaceof the bottom plate 102B). In other embodiments, the upper surface 301of the reservoir region can be defined by the bottom surface of themicrofluidic chip 106. For example, the reservoirs can be exposed andformed on the top surface of the bottom plate 102B. The exposedreservoirs can further become enclosed and covered when the microfluidicchip 106 is inserted into the chip pocket 312. With this implementation,the bottom surface of the microfluidic chip can correspond to the topsurface 301 of the reservoir region. Other configurations areenvisioned.

Each of these reservoirs (e.g., the buffer fluid reservoir 304, thewaste fluid reservoir 306, and the detection fluid reservoir 308) caninclude one or more openings through which the corresponding fluid caninjected into the reservoir and one or more openings through which fluidis removed from the reservoir. For example, as shown in FIG. 3B, each ofthe reservoir regions can include a single fluidic connection or openingthrough which fluid is injected from the bottom plate 102B and into thereservoir, or from which fluid is removed from the reservoir and ejectedthrough the bottom plate 102B. In particular, the buffer fluid reservoir304 can include fluidic connection 304′ through which buffer fluid canbe injected into the buffer fluid reservoir 304. Waste fluid reservoir306 can include fluidic connection 306′ through which waste fluid can beextracted from the waste fluid reservoir 306. Detection fluid reservoir308 can include fluidic connection 308′ through which detection fluidcan be inserted into the detection fluid reservoir 308. For example,with reference to FIGS. 3A and 3B, in some embodiments, inlet port 114can be configured to receive and facilitate injection of the bufferfluid into the buffer fluid reservoir 304 via a microfluidic channel(not shown) formed within the bottom plate 102B that connects the inletport 114 to the microfluidic connection 304′. In some implementations,an o-ring or another suitable gasket material can form a fluid seal atthe interface of the fluidic connection 304′ and the fluidic inletconnected thereto. Similarly, inlet port 116 can be configured toreceive and facilitate injection of the detection fluid into thedetection fluid reservoir 308 via a microfluidic channel (not shown)formed within the bottom plate 102B that connects the inlet port 116 tothe microfluidic connection 308′. In some embodiments, the solid lines318 that separate the reservoirs can be or correspond to o-rings. Withthese embodiments the o-rings do not seal the fluid at the interface ofthe fluidic connections 308′ and 304′. Rather, they corral the fluidwithin the respective reservoirs. In addition, the outlet port 118 canbe configured to receive and facilitate removal of waste fluid that iscollected in the waste fluid reservoir 306 (e.g., as injected from theseparation unit of the microfluidic chip into the waste fluid reservoir306) via a microfluidic channel (not shown) formed within the bottomplate 102B that connects the oulet port 118 to the microfluidicconnection 306′. In some implementations, an o-ring or another suitablegasket material can form a fluid seal at the interface of the fluidicconnection 308′ and the fluidic inlet connected thereto.

With reference to FIG. 3C in connection with reference to FIG. 2B, inone or more embodiments, the upper surface of the buffer fluid reservoir304 can align with and be fluidically connected to the plurality ofinput vias of the separation unit of the microfluidic chip 106. Forexample, the respective dashes of the dashed line 314′ depicted withinthe buffer fluid reservoir 304 can respectively correspond projectedinlet via locations of the separation unit through which the bufferfluid included in the buffer fluid reservoir 304 can be pushed to enterthrough aligned input vias (e.g., the second input vias described above)of the separation unit. With these implementations, the buffer fluid,can be injected into the buffer fluid reservoir 304 of the housing 102via inlet port 114 and fluidic connection 304′. As the buffer fluidreservoir 304 fills with buffer fluid, the buffer fluid can be pressuredriven through the aligned and fluidically connected second inlet viasof the separation unit 206. For example, in some embodiments, a pressuredriving device or system coupled to the separation-purificationapparatus 100 can be used to pressure drive the second fluid or bufferfluid through the inlet port 114 and/or fluid the buffer fluid reservoir304 and onto the microfluidic chip 106. In other implementations, thesecond fluid can be injected into inlet port 114 via a pipette, asyringe, or another suitable injection means. Similarly, the uppersurface of the waste fluid reservoir 306 can align with and befluidically connected to the plurality of outlet vias of the separationunit of the microfluidic chip 106 through which waste fluid is excreted.In this regard, the respective dashes of the dashed line 316′ depictedwithin the waste fluid reservoir 306 can respectively correspond toprojected outlet via locations of the separation unit through whichwaste fluid can be ejected into the waste fluid reservoir 106. Inaddition, a plurality of fluidic connections 212′ can be included in theupper surface of the detection fluid reservoir 308 which can befluidically connected to the one or more third inlet vias 212. Withthese implementations, the detection fluid reservoir 308 can befluidically coupled with the one or more third inlet vias 212 of themicrofluidic chip 106 through which the detection fluid can be injectedand flowed onto the sensing element 108 to facilitate coating thesensing 108 prior to flow of biological sample fluid through themicrofluidic chip 106. In this regard, as the detection fluid isinjected through inlet port 116 and fills the detection fluid reservoir308 via fluidic connection 308′, the detection fluid can evenly enterthe one or more third inlet vias 212 and evenly coat the sensing element108. For example, in some embodiments, the pressure driving device orsystem coupled to the separation-purification apparatus 100 can also beused to pressure drive the detection fluid through inlet port 116 and/orthe additional fluid reservoir and onto the microfluidic chip 106. Inother implementations, the third fluid can be injected into inlet port116 via a pipette, a syringe, or another suitable injection means.

With reference again to FIGS. 1A and 1B, the separation-purificationapparatus 100 can further include a sealing layer 104 formed between themicrofluidic chip 106 and the housing 102. For example, in theembodiment shown, the sealing layer 104 is provided between an uppersurface of the bottom plate 102B and a bottom surface or backside of themicrofluidic chip 106. In other implementations, the sealing layer 104can be formed at an interface between the reservoir region and the uppersurface of the bottom plate 102B. In yet another embodiment, the sealinglayer 104 can be formed at an interface between the reservoir region andthe bottom surface of the microfluidic chip.

The sealing layer 104 can comprise one or more materials that facilitatecreating a fluidic seal between one or more vias or openings in thebackside of the microfluidic chip 106, and one or more ports,capillaries, channels, and/or reservoirs of the bottom plate 102B and/orthe reservoir region. For example, in some implementations, the sealinglayer 104 can comprise a gasketing material that creates a fluidic sealat the interface between one or more openings or vias on the backside ofthe microfluidic chip 106, and one or more adjacent/aligned openings(e.g., a capillary, a channel, a via, a reservoir, etc.) in the uppersurface of the bottom plate 102B. For example, in some embodiments, thegasket material can be constructed from o-rings or a polymer, such as anelastomer, a thermoset, a thermoplast, and the like. In someimplementations, the gasket material can be formed by 3D printing,stamped, embossed, injection molded, laser cut, or ablated from thepolymeric starting material. For example, as can be observed bycomparing FIGS. 2B and 3B, the placement of the global and local viainlet and outlets on the chip surface are superimposed or projected ontocommon reservoirs, whose borders can be defined by sets of o-rings onthe backside of the chip, one means of providing a fluidic seal at theinterface between the chip and flow-cell/packing.

The sealing layer 104 can thus comprise one or more or more o-rings orgasket arrangement that ensure each reservoir of the housing and/ormicrofluidic chip is continuous, allowing one fluid type to beuniversally introduced or extracted from one associated via or set ofvias on the microfluidic chip 106. In this regard, although the sealinglayer 104 is depicted a continuous layer of material, it should beappreciated that this depiction is merely for exemplary purposes. Forexample, in implementations, the sealing layer 104 can consist of aplurality of o-rings formed in an arrangement only between portions ofthe interface between the microfluidic chip 106 and the housing 102where adjacent openings are located. This arrangement allows thesimultaneous fluid loading of all vias associated with a particularfluid group. Importantly, the fluid to/from each reservoir in the gasketmust have an inlet port or outlet port (e.g., one or more of ports112-120) that can be accessed from the housing 102. In the embodimentshown, only one access port to each reservoir is used to minimize thedesign complexity of the housing 102.

FIG. 4 illustrates an example cross-sectional view the detection region400 of separation-purification apparatus 100 taken along axis A-A′ shownin FIG. 1 in accordance with one or more embodiments described herein.Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

With reference to FIG. 4 in conjunction with FIGS. 1, 2A-2D and 3A-3C,in the embodiment shown, three vias or channels are formed within thethickness of the substrate (e.g., silicon) of the microfluidic chip 106,respectively corresponding two of the third inlet vias 212, and theglobal outlet via 210. In this regard, the inlet vias 212 respectivelyconnect to and/or correspond to respective third inlet vias 212 throughwhich detection fluid can be introduced onto the sensing element 108.Likewise, outlet via 210 connects to and/or corresponds to outlet via210 through which excess detection fluid and biological fluid sample canbe removed from the microfluidic chip. These channels are respectivelyfluidically coupled to and aligned with corresponding fluidicconnections 212′ and 310′ in the reservoir layer. For example, inletvias 212 are respectively connected to fluidic connections 212′ in thedetection fluid reservoir 308, and outlet via 210 is connected to thefluidic connection 310′ which connects to the outlet port 120. Inaccordance with this embodiment, the sealing layer 104 consists oro-rings respectively formed between the bottom surface of themicrofluidic chip 106 and the upper surface of the reservoir layer(e.g., included in the bottom plate 102B) around the aligned channelsvias 212 and 210 and their corresponding fluidic connections in thereservoir layer.

With reference again to FIG. 1, in various embodiments, although notshown, separation-purification apparatus 100 can be coupled to apressure-driving system to control flow of the various fluids (e.g., thebiological sample fluid, the buffer fluid, the detection fluid, andother potential cleaning/preparation fluids) into, out of, and throughthe housing 102 and the microfluidic chip 106. The pressure-drivingsystem can be a fully automated pressure driving machine, a manuallyoperated pressure driving machine, or a combination thereof. Thepressure driving system can form a pressure seal between the inlet ports(e.g., inlet port 112, inlet port 114, and inlet port 116) and therespective reservoirs, channels, inlets, etc., of the housing 102,and/or the respective outlet ports (e.g., outlet port 118 and outletport 120). In this regard, the pressure driving system can applypressure to initiate fluid flow of one or more fluids within thedifferent fluid reservoirs located within the housing and a reservoircomprising the biological sample fluid, referred to herein as the samplefluid reservoir. Although not explicitly shown in FIG. 3B, in someimplementations, the housing 102 can include the sample fluid reservoirformed therein in which the biological sample fluid can be pre-loadedprior to running through the microfluidic chip 106. In otherimplementations, the sample fluid reservoir can be provided external toseparation-purification apparatus 100. The sample fluid reservoir can befluidically coupled to the global inlet via 202 by way of the (gasketed)opening 302 of the housing 102 aligned therewith.

The separation-purification apparatus 100 can further include a cappinglayer 110 formed on or over the microfluidic chip 106. In variousembodiments, the capping layer 110 can include a transparent orsemi-transparent material (e.g., glass, plastic, etc.) that provides forhermetically sealing one or more fluidic elements (e.g., busses,channels, vias, reservoirs, sensing element 108 chambers, etc.),provided on or within the microfluidic chip 106. In addition, byemploying a transparent capping layer 110, the upper surface of themicrofluidic chip including the sensing element 108 can be visuallyobserved (e.g., via the naked eye, a microscope, or another suitableimaging device).

In some embodiments, the microfluidic chip 106 can be permanently sealedwithin the housing 102. With these embodiments, formation ofseparation-purification apparatus 100 can include a bonding procedurewherein the backside of the microfluidic chip 106 is bonded to orotherwise affixed to the upper surface of the bottom plate 102B of thehousing. The capping layer 110 can further be bound to the microfluidicchip 106 and/or the housing 102 to permanently seal the microfluidicchip 106 within the housing 102. In other embodiments, the microfluidicchip 106 can be removably attached to the housing 102. With theseembodiments, the housing 102 can be re-used with new microfluidic chipsinserted therein (or cleaned microfluidic chips reinserted therein). Forexample, in one implementation, the housing 102 can be configured toreceive and snap-in, screw in, lock-in, etc., the microfluidic chip 106in a manner that allows for the chip to be easily removed after use. Thecapping layer 110 can further be configured to removably attach or openand close to cover and seal the microfluidic chip 106 within the housing102 during use. Alternatively, the capping layer 110 can be permanentlyaffixed to the surface or the microfluidic chip 106. With thisimplementation, the microfluidic chip 106 with the capping layer 110bound thereto can for a single unit that can be inserted into thehousing 102. With these implementations, the top plate 102A of thehousing can be attached to the bottom plate 102B of the housing (e.g.,via one or more screws or another mechanism) to sandwich and seal themicrofluidic chip 106/capping layer 110 unit therein.

In accordance with an example usage scenario in whichseparation-purification apparatus 100 is used for biomarker discoverand/or liquid biopsy screening, separation-purification apparatus 100can be operated as follows. Optionally, the microfluidic chip 106 can bepre-wet with antifouling chemical agents including, but not limited tobuffers of varying pH and ionic strength levels, surfactants, andbiological coating agents such as bovine serum albumin (BSA). Next, asample (including urine, blood, plasma, saliva, cell culture media,etc.), buffer, and antibody or aptamer containing chemistries can beloaded (e.g., via pipetting or another suitable mechanism) into separatereservoirs of the housing 102 (e.g., located on or within the bottomplate 102B) with the microfluidic chip 106 sealed inside. The housingcan further be being placed into or otherwise coupled to a pressuredriven system to initiate purification and detection. Initially, thepressure-driven system can apply pressure to force the flow of theantibody or aptamer containing fluid onto the sensing element 108 tosurface functionalize the sensing element 108. The pressure-drivensystem can then stop the flow of antibody or aptamer chemistry andinitiate the flow of sample and buffer simultaneously to initiatepurification and downstream immunocapture of target macromolecules thatflow over the sensing element 108. The presence of one or more exosomesor other biomarkers bound to the antibodies or aptamers coated on thesurface of the sensing element 108 can be observed either directly by anoperator (e.g., via the naked eye, through a microscope lens placed overthe sensing element 108, etc.) or computer-based reception and analysisof image data captured of the sensing element 108.

FIG. 5 illustrates an enlarged view of an example separation unit 206 ofthe microfluidic chip 106 in accordance with one or more embodimentsdescribed herein. For example, call-out box 501 presents and enlarged orzoomed-in view of the area of the microfluidic chip 106 encircled by box500. Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

With reference to call-out box 501, the separation unit 206 can comprisea plurality of DLD on nanoDLD arrays formed between inlet bus 204 andoutlet bus 208. In this regard, the size and spacing of the respectivepillars used for DLD array can be adapted to a particular targetparticle size or size range, which can include nanoparticles, such asexomes, viruses, DNA sequences, RNA sequences, etc., as well as largerparticles such as cells. Thus, in implementations in which themicrofluidic chip 106 is used for exosome filtration and biomarkeranalysis, the separation unit 206 can employ nanoDLD arrays. For largerparticles, the separation unit 206 can employ DLD arrays with pillarsand/or pillar spacing adapted to filter particles greater than 999 nm.For ease of explanation, the DLD array portion of the separation unit206 is referred to as a nanoDLD array 506.

The separation unit 206 can employ a multiplexed arrangement of aplurality of conjoined nanoDLD array pillars or units to facilitateenhanced throughput of target particle isolation from sample biologicalfluid. For example, with reference to FIG. 2B and FIG. 5, the separationunit 206 can employ four rows of densely packed nanoDLD array pairsarranged inside of the circular, inlet bus 204 feed that distributes thesample fluid from the global inlet via 202. For example, the number ofnanoDLD array pairs that can be integrated into the microfluidic chipcan range from the thousands to the hundreds of thousands depending onthe size of the chip. Usage of this massive, multiplexed parallelizationframework can substantially increase the throughput rate of theseparation unit 206 (e.g., to around 1.0 mL of sample fluid per hour orgreater).

The nanoDLD array 506 can include alternating pillar array units withdifferent pillar angles for outward deflection of an incoming samplefluid, and inward deflection for incoming buffer fluid. For example, inthe embodiment shown, the separation unit 206 can include a plurality ofsecond inlet vias 502 through which buffer fluid can be introduced intothe separation unit 206 and flowed downstream (e.g., in the direction ofarrow D) in association with simultaneous flow of biological samplefluid through the separation unit 206 from inlet bus 204 (e.g., in thedirection of arrow D). As described with reference to FIGS. 2B and 3C,for example, the second inlet vias 502 can correspond to the respectivedark boxes of the first checkered line drawn at the interface betweenthe inlet bus 204 and the dark grey region of the separation unit 206.The second inlet vias 502 can further connect to the buffer fluidreservoir 304.

A zoomed-in view of the nanoDLD array pairs is shown in call out-boxes503 and 505. As shown in call-out box 501 and call-out box 503, thenanoDLD array 506 can include outward deflection units 506 a positionedadjacent to the entry region of the separation unit 206 with thebiological fluid sample is passed from the inlet bus 204. As shown incall-out box 501 and call-out box 505, the nanoDLD array 506 can furtherinclude inward deflection units 506 b positioned adjacent to the secondinlet vias 502 through which the buffer fluid is injected. As a result,larger, target particles of interest included in the biological fluidsample can be directed in an inward flow path direction that causes afirst stream of buffer fluid including the particles of interest to flowtoward and into the outlet bus 208 where they are collected. Smaller,unwanted particles (e.g., salts, proteins, lipids, and other smallbiomolecules/macromolecules), collectively referred to herein as wasteparticles, can be directed in an outward flow path direction that causesa second stream of buffer fluid to flow toward and into a plurality ofoutlet vias 508. For example, as described with reference to FIGS. 2Band 3C, the outlet vias 508 can correspond to the respective dark boxesof the second checkered line drawn at the interface between the darkgrey region of the separation unit 206 and the outlet bus 208. Theoutlet vias 508 can further align with and connect to the waste fluidreservoir 306. In this regard, the portion of the buffer streamincluding the waste particles can flow into the waste fluid reservoir306 via the outlet vias 508 where the waste fluid can be collected andfurther exported off the chip and the housing 102 (e.g., through outletport 118).

In the embodiment shown, the separation unit 206 can also include afiltration element 504 (depicted by the plurality of speckles or dots)provided at or near the initial entry points of the buffer fluid and thesample fluid into the separation unit 206. For example, the filtrationelement 504 can be formed upstream of the nanoDLD array 506. Thefiltration element 504 can (optionally) be used to filter out or removecertain large particles of size greater than a defined threshold size,such as a size greater than the target particles (e.g., greater thanexosomes). In one or more embodiments, the filtration element caninclude but is not limited to, a cross-flow of serpentine filters,traps, sieves, bladed loading features or a number of other microfluidicfilter arrangements to capture cells, larger cellular debris, and/orlarger multivesicular bodies (MVBs) while letting desired colloids passinto the downstream, nanoDLD array 506.

FIG. 6 illustrates another enlarged view of the portion of themicrofluidic chip 106 included in call-out box 401. FIG. 6 furtherincludes call-out box 600 depicting a nano-scale view of one of theoutward deflection units 506 a in association with simultaneous flow ofsample biological fluid and buffer fluid through the nanoDLD arrays inaccordance with one or more embodiments described herein. Repetitivedescription of like elements employed in respective embodiments isomitted for sake of brevity.

FIG. 6 highlights an example process of operation for the separationunit 206 of example separation-purification apparatus 100. In theembodiment shown, the separation process of the separation-purificationapparatus 100 involves inlet bus 204 for introduction of sample fluid,second inlet vias 502 through which buffer fluid is injected, filtrationelement 504, nanoDLD array 506, outlet vias 508 for excreting wastefluid, and outlet bus 208 for collecting purified or extracted particlesof interest (e.g., exosomes) and carrying the particles of interest tothe downstream detection unit. In this regard, with reference to FIGS.2B, 3B and FIG. 6, a sample fluid, such as plasma, cultured medium,urine, etc., can be introduced into the microfluidic chip 106 (e.g., atglobal via 202), and fed through the (circular) inlet bus 204, which inturn, injects the sample through openings at the interface of the inletbus 204 and the separation unit 206. For example, these openings arelocated adjacent to the outward deflection units 506 a of the nanoDLDarray 506. Buffer fluid is further simultaneously injected into theseparation unit 206 through the second inlet vias 502 adjacent to theinward deflection areas of the nanoDLD arrays. The buffer fluid canprovide a fresh purification medium. Prior to flow of the sample fluidthrough the nanoDLD array 506, the filtration element 504 can removelarger particles of material from the incoming sample fluid that mightotherwise clog at the interface of nanoDLD arrays, reducing longevity.

As shown in FIG. 6, as the fluid sample and the buffer fluidsimultaneously flow through the nanoDLD array 506, the large targetparticles of a defined size range (e.g., exosomes having a defined sizerange), are deflected or bumped into a portion of the buffer fluid whichgets focused at the inward deflection unit/outward deflection unitjunction. As a result, the large target particles are directed into theoutlet bus 208. Smaller unwanted particles, such as salts, smallmolecules, lipids, proteins, etc. maintain their general trajectory inthe direction of the sample fluid flow toward the outlet vias 508, alsoreferred to herein as the waste outlets. Through this process, aparticular purified size range of target particles (e.g., exosomes) canbe selectively loaded into the outlet bus 208. This purificationprocess, in effect, acts as a bandpass filter to remove the backgroundcontamination below and larger material above structurally definedcutoffs, making it much more straightforward to detect target particlesbearing a particular surface marker or biomarker on the downstream,sensing element 108.

FIG. 7 illustrates an enlarged view of an example detection unit 700 ofan example microfluidic chip (e.g., microfluidic chip 106) thatintegrates on-chip particle purification and biomarker detectionfunctionality in accordance with one or more embodiments describedherein. Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

The detection unit 700 can include the sensing element 108. In theembodiment shown, the detection unit 700 can also include the portion ofthe outlet bus 208 that connects to the sensing element 108 and providesfor injecting a stream of buffer fluid comprising the purified orseparated target particles onto and over the surface of the sensingelement 108 coated with a surface chemistry (e.g., one or more specificmolecules or macromolecules) that provides chemical specificity for oneor more known biomarkers that may be present on or within the targetparticles. In this regard, the as discussed above, the sensing element108 can facilitate detecting presence of one or more biomarkers bycoating or otherwise providing one or more reactive agents that have aknown chemical specificity for the one or more biomarkers, such asantibodies and aptamers known to bind with one or more surface markers.In one or more example implementations, the sensing element 108 caninclude one or more antibodies or aptamers known to bind with exosomesbearing a particular oncogenic surface marker (e.g. CD81, PSMA, etc.).The chemical specificity however is not limited to antibody/epitopeinteractions but can be extended to any specific chemical or biochemicalinteraction between two molecules or macromolecules, including naturallyoccurring and synthetic molecules/macromolecules. For example, thechemical interactions can include permanent covalent linkage as well asreversible bond interactions, such as electrostatic interactions,hydrophobic interactions, complementarity interactions and the like. Inthis regard, the sensing element 108 can provide for detectingbiomarkers are result of interactions including but not limited to,antibody-epitope interactions, complementary DNA or RNA strandhybridization interactions, DNA binding proteins and DNA consensussequence interactions, protein-protein interactions, protein-smallmolecule interactions, polymerization reactions, biotin-streptavidininteractions, and others.

The sensing element itself 108 can take on a variety of structures andmaterials to enhance the signal generated as a result of a chemicalreaction between the surface chemistry of the sensing element and theone or more biomarkers. For example, the sensing element 108 can includean optical element that enhances a visual signal generated or detectedin association with binding of a antibody provided on the surfaced ofthe sensing element with an epitope of a target particle, such asfluorescence signal of immuno-bound exosomes, thereby enhancing thesensitivity of the sensing element, In this regard, the sensing element108 can include but is not limited to, a photonic grating or pillararray, an optoelectrical element, or a plasmonic structure.

The detection unit 700 can further include one or more third inlet vias212 through which detection fluid including the reactive analytesubstances can be introduced and applied to coat the surface of thesensing element 108. The detection unit 700 can also include adistribution bus 214 to facilitate evenly coating the sensing elementwith the introduced detection fluid. For example, in one or moreembodiments, prior to introducing purified sample to the sensing element108, the sensing element 108 can be coated with appropriate detectionmolecules/macromolecules (e.g., antibodies, aptamers, etc.) inaccordance with a coating process (noted in FIG. 7 as coating step 1).With reference to FIGS. 2C-2D, FIG. 3C and FIG. 7, in accordance withthe coating process, detection fluid (e.g., antibody-containing fluid)can be introduced through the one or more third inlet vias 212 byapplying a positive pressure at these inlets, or more directly to thedetection fluid reservoir 308 to which the one or more third inlet vias212 are fluidically connected (e.g., via fluidic connections 212′). Atthe same time, a comparatively negative pressure can be applied at theglobal outlet via 210. The binding chemistry of the detection fluid canthus be forced to flow from one or more third inlet vias 212 to theglobal outlet via 210, thereby coating the sensing element 108. In someimplementations, the sensing element can be pre-wetted prior to thecoating step to facilitate the coating process. Once the sensing element108 has been coated the separation-purification apparatus 100 is readyto use particle purification and separation. In this regard, inaccordance with step 2, the upstream separation can then be performed inaccordance with the techniques described herein, bringing the purifiedparticles (e.g., exosomes) to the sensing element 108 by way of theoutlet bus 208, where they can potentially bind to the sensing element108 (if a particular target exosome and/or biomarker is present) andgenerate a reactionary signal that can be detected. For example, in someimplementations, a target particle can be detected throughimmunocapture, producing a fluorescent signal that can be observed withfluorescence microscopy, and therefore manually detectable by eye orthrough software to automate the process.

FIG. 8 illustrates an enlarged view of another example detection unit800 of an example microfluidic chip (e.g., microfluidic chip 106) thatintegrates on-chip particle purification and biomarker detectionfunctionality in accordance with one or more embodiments describedherein. Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

Detection unit 800 can include same or similar features andfunctionality as detection unit 700 with the addition of a blockingelement 802 at the inlet interface between the one or more third inletvias 212 and the sensing element 108, (e.g., including openings wherethe detection fluid can flow from the one or more second inlet vias ontothe surface of the sensing element 108. The primary function of theblocking element 802 is to block free floating, unbound or unreactedtarget particles (e.g., exosomes), from reversely flowing away from thesensing element 108 and the global outlet via 210 toward the one or morethird inlet vias 212. In some implementations, in which detectionmolecules are not tethered to or become separated from the surface ofthe sensing element, the blocking element 802 can also prevent reverseflow of free floating reacted molecular complexes (e.g.,antibody-exosome complexes) through the third inlet vias 212. In thisregard, the blocking element 802 can include a physical structure thatprevents essentially any particles other than free floating detectionmolecules/macromolecules (which are generally very small) therethrough.The blocking element 802 can thus corral isolated target biologicalentities (e.g. exosomes, viruses, etc.) with biochemically specificmarkers within the field of view of an optical detector. For example, asshown in call out boxes 801 and 803, in one embodiment, the blockingelement 802 can comprise integrated pillars, a sieve, or the like, withgaps in between them at the perimeter of the sensing element 108 andadjacent to the detection fluid chemistry inlets. The gaps can be toosmall for free-flowing isolated target particles (e.g., exosomes) to fitthrough, but large enough for the coating chemistry to flow through(e.g., antibodies in the example shown). In this way, the blockingelement can prevent loss of exosomes bearing the target surfaceproteins, or epitopes, keeping them contained completely within thefield of view and surface of the sensing element 108.

FIG. 9A illustrates an enlarged view of another example detection unit900 that can be employed in a microfluidic chip (e.g., microfluidic chip106) that integrates on-chip particle purification and biomarkerdetection functionality in accordance with one or more embodimentsdescribed herein. Detection unit 900 can include same or similarfeatures and functionality as detection unit 800, with the addition of aplurality of different detection chambers to the sensing element 108.Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

In the embodiment shown, the sensing element 108 can be subdivided intoa plurality of separate detection chambers, respectively identified asdetection chambers 901, 902, 903 and 904. Each of the differentdetection chambers can provide for detecting a presence of differentbiomarker and/or target particle. For example, each (or in someimplementations one or more) of the detection chambers 901, 902, 903 and904 can be coated with different detection molecules/macromoleculesknown to react with different biomarkers. For instance, the respectivedetection chambers can be coated with different biomarker-specificantibodies or aptamers. With these embodiments, the sensing element 108can provide for simultaneous detection of a plurality of potentialbiomarkers from a single input fluid sample and with a singlepurification-detection procedure. Simultaneous detection of multiplemarkers allows for fast, and effective diagnosis of various diseases,such as certain forms of cancer. In order to ensure isolation betweenthe different detection chambers, the detection chambers canrespectively be separated from one another via partition wall 906.

In accordance with this embodiment, a separate detection fluid should beinjected into each of the respective detection chambers in isolation. Inthis regard, each of the detection chambers can employ a different inletvia (of the one or more third inlet vias 212) that can respectively befluidically coupled to different detection fluid reservoirs (e.g., asopposed to the single, communal, detection fluid reservoir 308, shown inFIGS. 3A-3C). For example, as shown in FIG. 9B, rather than a communal,detection fluid reservoir 308, reservoir layer (included in the bottomplate 102B or between the bottom plate 102B and the microfluidic chip106) can include a plurality of isolated detection fluid reservoirs orchannels 910 through which the different detection fluids can beinjected to coat the different detection chambers.

FIGS. 9C and 9D present a 3D, perspective view of example detection unit900 in accordance with one or more embodiments described herein. Withreference to FIGS. 2C and 2D in conjunction with reference to FIGS. 9Cand 9D, detection unit 900 can include same or similar features andfunctionalities as detection unit 200 with the addition of separatedetection chambers 901, 902, 903 and 904 to the sensing element 108. Therespective detection chambers are separated by separation walls 906. Inaddition, a blocking element 802 is formed at the inlet region betweenthe respective third inlet vias 212 and the sensing element 108. Asshown in FIG. 9D with reference to the dashed arrow lines, after thedifferent detection chambers of the sensing element 108 have beenfunctionalized with a different surface chemistry, a stream of bufferfluid comprising purified target particles can flow from the separationunit 206, up through the outlet bus 208 channels and onto the sensingelement 108. In the embodiment shown, a single outlet bus 208 channelcan feed two detection chambers at a time (e.g., detection chambers 903and 904 in the demonstrated example).

FIG. 10 illustrates an enlarged view of another example detection unit1000 that can be employed in a microfluidic chip (e.g., microfluidicchip 106) that integrates on-chip particle purification and biomarkerdetection functionality in accordance with one or more embodimentsdescribed herein. Detection unit 1000 can include same or similarfeatures and functionalities as detection unit 900 with the addition ofa greater number of detection chambers, including detection chambers1001-1008. In this regard, the number of detection chambers in which thesensing element is subdivided into can vary and is not limited to one,four or eight (as in the embodiments shown). However, as the number ofdetection units increases, the number of third inlet vias 212 will alsoincrease, as each detection unit can comprise a separate injection via(and corresponding channel and/or reservoir) through which eachdifferent type of detection fluid chemistry can be provided. Repetitivedescription of like elements employed in respective embodiments isomitted for sake of brevity.

FIG. 11 illustrates an example system 1100 that facilitates integratingreal-time particle purification and biomarker detection in accordancewith one or more embodiments described herein. Embodiments of systems(e.g., system 1100 and pressure driving system 1102), imaging devices(e.g., imaging device 1104) and computing devices (e.g., computingdevice 1108) described herein can include one or more machine-executablecomponents embodied within one or more machines (e.g., embodied in oneor more computer readable storage media associated with one or moremachines). Such components, when executed by the one or more machines(e.g., processors, computers, computing devices, virtual machines, etc.)can cause the one or more machines to perform the operations described.Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

System 1100 includes separation-purification apparatus 100, a pressuredriving system 1102 an imaging device 1104 and a computing device 1108.The computing device 1108 can be communicatively coupled to the imagingdevice 1104 and/or the pressure driving system 1102 via one or morewires and/or one or more wireless networks (e.g., a local area network(LAN), a wide area network (WAN), such as the Internet, and the like).

The pressure driving system 1102 can be operatively coupled to one ormore inlet ports and outlet ports of the housing (e.g., which can be orcorrespond to a microfluidic flow cell or the like) to control flow ofthe various fluids (e.g., the biological sample fluid, the buffer fluid,the detection fluid, and other potential cleaning/preparation fluids)into, out of, and through the housing 102 and the microfluidic chip 106.For example, in some implementations, after the above noted fluids areintroduced into the designated reservoirs of the housing 102 (e.g., viapipetting or another suitable technique), the apparatus can beoperatively coupled to the pressure driving system 1102 and while alsobeing aligned with the imaging device 1104. In some implementations, theimaging device 1104 and the pressure driving system 1102 can be acombined system. In other implementation, the pressure driving system1102 can facilitate injecting the various fluids into the correspondingreservoirs of the housing 102 prior to and/or at runtime of theapparatus. The pressure-driving system can be a fully automated pressuredriving machine, a manually operated pressure driving machine, or acombination thereof. The pressure driving system can form a pressureseal between the inlet ports (e.g., inlet port 112, inlet port 114, andinlet port 116) and the respective reservoirs, channels, inlets, etc.,of the housing 102, and/or the respective outlet ports (e.g., outletport 118 and outlet port 120). In this regard, the pressure drivingsystem can apply pressure to initiate fluid flow of one or more fluidswithin the different fluid reservoirs located within the housing and areservoir comprising the biological sample fluid, referred to herein asthe sample fluid reservoir.

The imaging device 1104 can comprise a lens 1006 or capture region thatis aligned with the sensing element 108. In this regard, the location ofthe sensing element 108 at or near the center of the microfluidic chip106 (with a transparent window formed thereover as part of the cappinglayer 110), can enable efficient optical readout of biochemicallyspecific information that developed or captured by the sensing element(e.g., immunocaptured exosomes detected using for example fluorescencemicroscopy). In the embodiment shown, the imaging device 1104 is amicroscope. In some implementations in which the imaging device 1104comprises a microscope, the microscope can be or include a fluorescencemicroscope configured to capture immunofluorescent signals generated byfluorescent labeled molecules (e.g., antibodies or aptamers, targetparticles, binding proteins, etc.) captured by the sensing element 108.The features and functionalities of the microscope can however vary. Theimaging device 1104 can alternatively or additionally include othertypes of imagining devices or cameras configured to captures stillimages, 2D images, high dynamic range images, video, etc. of the sensingelement 108 at various stages of operation of separation-purificationapparatus 100 in accordance with the techniques described herein (e.g.,in real-time during separation and detection flow or after completion ofrunning of the biological fluid sample therethrough). In someimplementations, these images can be sent to a computing device 1108 forrendering via a display 1110 of the computing device 1108. In thisregard, in some implementations, biomarker detection and/or biochemicalanalysis of target particles with specific surface markers (e.g.,exosomes with specific carcinogenic surface markers) can be manuallyperformed by examining the sensing element 108 through the microscopeand/or via image data presented via the display 1110 in real-time duringseparation and detection flow, or after completion of running of thebiological fluid through the apparatus. In other implementations,described infra, the computing device 1108 can include softwareconfigured to perform automated biomarker detection and analysis basedon image data captured of the sensing element 108 before, during, and/orafter running of biological fluid sample through separation-purificationapparatus 100.

FIG. 12 illustrates an example computing device 1108 that facilitatesreal-time biomarker detection and analysis in accordance with one ormore embodiments described herein. Repetitive description of likeelements employed in respective embodiments is omitted for sake ofbrevity.

As presented in system 1100, the computing device 1108 can include or beoperatively coupled to a display 1110 via which image data captured ofthe sensing element 108 can be rendered. The computing device 1108further includes or can be operatively coupled to at least one memory1212 and at least one processor 1210. In various embodiments, the atleast one memory 1212 can store executable instructions that whenexecuted by the at least one processor 1210, facilitate performance ofoperations defined by the executable instruction. For example, in theembodiment shown, the computing device 1108 further include microfluidicsensor processing module 1202 which includes sensor data receptioncomponent 1204, biomarker detection component 1206, and diagnosiscomponent 1208. In one or more embodiments, these components (e.g., themicrofluidic sensor processing module 1202 and additional components ofthe microfluidic sensor processing module 1202) can be stored in memory1212 and executed by the at least one processor 1210. The computingdevice 1108 can further include a device bus 1214 that communicativelycouples the various components of the computing device 1108 (e.g., themicrofluidic sensor processing module 1202, the processor 1210, thememory 1212 and the display 1110). Examples of said processor 1210 andmemory 1212, as well as other suitable computer or computing-basedelements, can be found with reference to FIG. 16, and can be used inconnection with implementing one or more of the systems or componentsshown and described in connection with FIGS. 11 and 12 or other figuresdisclosed herein.

The microfluidic sensor processing module 1202 can facilitate variousprocessing functionalities associated with evaluating chemical reactionsthat occur (or do not occur) at the sensing element 108 of the disclosedmicrofluidic chips. In this regard, the sensor data reception component1204 can receive data regarding the chemical reaction that occur (or donot occur) at a particular sensing element in association with flow ofpurified target biological entities over the surface of a functionalizedsensing element. In various embodiments, this data can include imagedata captured of the sensing unit before, during, and/or after flowprocess. For example, the image data can include still images of thesensing element 108 captured by an imaging device (e.g., imaging device1104) positioned in line-of-sight of the sensing element at one or morepoints before, during and/or after the flow process. In otherimplementations, the image data can include video captured during theflow process by such an imaging device. With these implementations, themicrofluidic sensor processing module 1202 can provide for real-time orlive biomarker detection and analysis. In some embodiments, the chemicalreactions that occur at the sensing element 108 between a targetparticle or target biomarker and the one or more detectionmolecules/macromolecules with which the sensing element 108 isfunctionalized can result in other forms of detectable sensory data,other than visual signals. For example, in some implementations, achemical reaction can be detected by generation of a detectableelectrochemical signal, generation a heat signal, or another form ofsensory data. With these implementations, the sensor data receptioncomponent 1204 can receive information regarding generation of suchother types of sensory signals to facilitate biochemical analysis.

The biomarker detection component 1206 can analyze received sensory data(e.g., image data, or another form of sensory data) captured of and/orgenerated at the sensing element regarding occurrence, (ornon-occurrence), of one or more chemical reactions between one or moreparticles in the purified biological sample stream and one or moredetection molecules/macromolecules of the sensing element 108, anddetermine whether a particular biomarker is present. In someimplementations, the biomarker detection component 1206 can alsodetermine a quantitative measure of the amount of detected biomarker. Inthis regard, the biomarker detection component 1206 can access and/oremploy biomarker identification information (e.g., stored in memory)that correlates potential chemical reaction-based image signals that canbe generated at a sensing element (e.g., based on the type of detectionmolecules/macromolecules with which the sensing element isfunctionalized), with known biomarkers and/or known particles. Thebiomarker detection component 1206 can further be configured to identifyor otherwise recognize an image signal that correlates with a knownbiomarker or particle to determine whether the biomarker is present. Forexample, in some implementations, the image signals can include imagedata such, as fluorescent image data, that visually tags a chemical bondbetween a target biomarker or surface marker and a detectionmolecule/macromolecule. In other implementation, the image signal datacan include a particular coloration or change in coloration, aparticular brightness or change in brightness, a particular imagepattern, and the like. In various embodiments, based on the analysis ofthe received sensory data (e.g., image data) the biomarker detectioncomponent 1206 can generate biomarker information (for rendering via thedisplay or for otherwise providing to an entity via a suitable outputdevice) identifying detected biomarkers and/or particles, and in someimplementations, an amount of the detected biomarkers and/or particles.

The diagnosis component 1208 can further analyze biomarker informationto determine diagnosis information regarding a disease state or medicalcondition of the entity (e.g., a patient) from which the biologicalfluid sample was taken. In this regard, the diagnosis component 1208 canaccess and employ information that correlates known biomarkers, knownbiomarker amounts, and/or known biomarker combinations (inimplementations in which two or more biomarkers can be detected at atime, such as with respect to detection unit 200, detection unit 800,detection unit 900, detection unit 1000 and the like), with particulardiseases, disease states, and/or medical conditions, to determinewhether a disease, disease state, and/or medical condition has beendetected.

FIG. 13 illustrates a flow diagram of an example, non-limiting method1300 for performing particle purification and biomarker detection usingan integrated microfluidic device in accordance with one or moreembodiments described herein. Repetitive description of like elementsemployed in respective embodiments is omitted for sake of brevity.

At 1302, target biological entities having a defined size range areisolated from other biological entities included in a biological fluidsample using a separation unit (e.g., separation unit 206) comprisingone or more nanoDLD arrays formed on a microfluidic chip (e.g.,microfluidic chip 106), thereby resulting in isolated target biologicalentities. At 1304 buffer fluid comprising the isolated target biologicalentities is driven through a conduit (e.g., outlet bus 208) of themicrofluidic chip from the separation unit to a sensing element (e.g.,sensing element 108) formed on the microfluidic chip. At 1306, detectionof the presence of one or more biomarkers associated with the isolatedtarget biological entities is facilitated based on whether a detectablesignal is generated at the sensing element in response to the driving.

FIG. 14 illustrates a flow diagram of an example, non-limiting method1400 for functionalizing a sensing element of an integrated microfluidicdevice and thereafter, employing the integrated microfluidic device toisolate exosomes and detect presence of exosomal surface markers basedon reaction with the functionalized sensing element, in accordance withone or more embodiments described herein. Repetitive description of likeelements employed in respective embodiments is omitted for sake ofbrevity.

At 1402, functionalizing a surface of a sensing element (e.g., sensingelement 108) of a microfluidic chip (e.g., microfluidic chip 106) withone or more detection molecules or macromolecules (e.g., antibodies,aptamers, etc.) that chemically react with one or more exosomal surfacemarkers, wherein the functionalizing comprises injecting a solutioncomprising the one or more detection molecules or macromolecules into achamber enclosing the surface of sensing element via at least oneinjection inlet (e.g., the one or more third inlet vias 212) of themicrofluidic chip. At 1404, exosomes are isolated from other biologicalentities included in a biological fluid sample in response to drivingflow of the biological fluid sample and a buffer fluid through amultiplexed nanoDLD array (e.g., nanoDLD array 506) of the microfluidicchip. At 1406, a stream of the buffer fluid comprising the exosomes isdriven from the multiplexed nanoDLD array (e.g., via outlet bus 208)over the surface of the sensing element and through an outlet via (e.g.,global outlet via 210) of the microfluidic chip located downstream ofthe sensing element.

FIG. 15 illustrates a flow diagram of an example, non-limiting method1500 that facilitates integrating real-time particle purification andbiomarker detection in accordance with one or more embodiments describedherein. Repetitive description of like elements employed in respectiveembodiments is omitted for sake of brevity.

At 1502, target biological entities having a defined size range fromother biological entities included in a biological fluid sample areisolated using a separation unit comprising one or more nanoDLD arrays(e.g., separation unit 206), thereby resulting in isolated targetbiological entities, wherein the separation unit is formed on amicrofluidic chip contained within a housing (e.g., housing 102). At1504, a buffer fluid comprising the isolated target biological entitiesis driven through a conduit of the microfluidic chip (e.g., outlet bus208) from the separation unit to a sensing element (e.g., sensingelement 108) formed on the microfluidic chip, wherein the sensingelement generates a visual signal in response to detection of presenceof one or more defined biomarkers associated with the target biologicalentities. At 1506, image data of the detection unit is captured inassociation with the driving (e.g., via imaging device 1104). At 1508, acomputing device comprising a processor (e.g., computing device 1108)determines a diagnosis regarding a medical condition or a disease stateassociated with the biological fluid sample based on analysis of theimage data (e.g., using biomarker detection component 1206 and/ordiagnosis component 1208).

In order to provide a context for the various aspects of the disclosedsubject matter, FIG. 16 as well as the following discussion are intendedto provide a general description of a suitable environment in which thevarious aspects of the disclosed subject matter can be implemented. FIG.16 illustrates a block diagram of an example, non-limiting operatingenvironment 1600 in which one or more embodiments described herein canbe facilitated. For example, the operating environment 1600 can compriseand/or otherwise facilitate one or more features of the pressure drivingsystem 1102, the imaging device 1104, and/or the computing device 1108described herein in accordance with one or more embodiments. Repetitivedescription of like elements employed in other embodiments describedherein is omitted for sake of brevity.

With reference to FIG. 16, a suitable operating environment 1600 forimplementing various aspects of this disclosure can include a computer1612. The computer 1612 can also include a processing unit 1614, asystem memory 1616, and a system bus 1618. The system bus 1618 canoperably couple system components including, but not limited to, thesystem memory 1616 to the processing unit 1614. The processing unit 1614can be any of various available processors. Dual microprocessors andother multiprocessor architectures also can be employed as theprocessing unit 1614. The system bus 1618 can be any of several types ofbus structures including the memory bus or memory controller, aperipheral bus or external bus, and/or a local bus using any variety ofavailable bus architectures including, but not limited to, IndustrialStandard Architecture (ISA), Micro-Channel Architecture (MSA), ExtendedISA (EISA), Intelligent Drive Electronics (IDE), VESA Local Bus (VLB),Peripheral Component Interconnect (PCI), Card Bus, Universal Serial Bus(USB), Advanced Graphics Port (AGP), Firewire, and Small ComputerSystems Interface (SCSI). The system memory 1616 can also includevolatile memory 1620 and nonvolatile memory 1622. The basic input/outputsystem (BIOS), containing the basic routines to transfer informationbetween elements within the computer 1612, such as during start-up, canbe stored in nonvolatile memory 1622. By way of illustration, and notlimitation, nonvolatile memory 1622 can include read only memory (ROM),programmable ROM (PROM), electrically programmable ROM (EPROM),electrically erasable programmable ROM (EEPROM), flash memory, ornonvolatile random access memory (RAM) (e.g., ferroelectric RAM (FeRAM).Volatile memory 1620 can also include random access memory (RAM), whichacts as external cache memory. By way of illustration and notlimitation, RAM is available in many forms such as static RAM (SRAM),dynamic RAM (DRAM), synchronous DRAM (SDRAM), double data rate SDRAM(DDR SDRAM), enhanced SDRAM (ESDRAM), Synchlink DRAM (SLDRAM), directRambus RAM (DRRAM), direct Rambus dynamic RAM (DRDRAM), and Rambusdynamic RAM.

Computer 1612 can also include removable/non-removable,volatile/non-volatile computer storage media. FIG. 16 illustrates, forexample, a disk storage 1624. Disk storage 1624 can also include, but isnot limited to, devices like a magnetic disk drive, floppy disk drive,tape drive, Jaz drive, Zip drive, LS-100 drive, flash memory card, ormemory stick. The disk storage 1624 also can include storage mediaseparately or in combination with other storage media including, but notlimited to, an optical disk drive such as a compact disk ROM device(CD-ROM), CD recordable drive (CD-R Drive), CD rewritable drive (CD-RWDrive) or a digital versatile disk ROM drive (DVD-ROM). To facilitateconnection of the disk storage 1624 to the system bus 1618, a removableor non-removable interface can be used, such as interface 1626. FIG. 16also depicts software that can act as an intermediary between users andthe basic computer resources described in the suitable operatingenvironment 1600. Such software can also include, for example, anoperating system 1628. Operating system 1628, which can be stored ondisk storage 1624, acts to control and allocate resources of thecomputer 1612. System applications 1630 can take advantage of themanagement of resources by operating system 1628 through program modules1632 and program data 1634, e.g., stored either in system memory 1616 oron disk storage 1624. It is to be appreciated that this disclosure canbe implemented with various operating systems or combinations ofoperating systems. A user enters commands or information into thecomputer 1612 through one or more input devices 1636. Input devices 1636can include, but are not limited to, a pointing device such as a mouse,trackball, stylus, touch pad, keyboard, microphone, joystick, game pad,satellite dish, scanner, TV tuner card, digital camera, digital videocamera, web camera, and the like. These and other input devices canconnect to the processing unit 1614 through the system bus 1618 via oneor more interface ports 1638. The one or more Interface ports 1638 caninclude, for example, a serial port, a parallel port, a game port, and auniversal serial bus (USB). One or more output devices 1640 can use someof the same type of ports as input device 1636. Thus, for example, a USBport can be used to provide input to computer 1612, and to outputinformation from computer 1612 to an output device 1640. Output adapter1642 can be provided to illustrate that there are some output devices1640 like monitors, speakers, and printers, among other output devices1640, which require special adapters. The output adapters 1642 caninclude, by way of illustration and not limitation, video and soundcards that provide a means of connection between the output device 1640and the system bus 1618. It should be noted that other devices and/orsystems of devices provide both input and output capabilities such asone or more remote computers 1644.

Computer 1612 can operate in a networked environment using logicalconnections to one or more remote computers, such as remote computer1644. The remote computer 1644 can be a computer, a server, a router, anetwork PC, a workstation, a microprocessor based appliance, a peerdevice or other common network node and the like, and typically can alsoinclude many or all of the elements described relative to computer 1612.For purposes of brevity, only a memory storage device 1646 isillustrated with remote computer 1644. Remote computer 1644 can belogically connected to computer 1612 through a network interface 1648and then physically connected via communication connection 1650.Further, operation can be distributed across multiple (local and remote)systems. Network interface 1648 can encompass wire and/or wirelesscommunication networks such as local-area networks (LAN), wide-areanetworks (WAN), cellular networks, etc. LAN technologies include FiberDistributed Data Interface (I-DDI), Copper Distributed Data Interface(CDDI), Ethernet, Token Ring and the like. WAN technologies include, butare not limited to, point-to-point links, circuit switching networkslike Integrated Services Digital Networks (ISDN) and variations thereon,packet switching networks, and Digital Subscriber Lines (DSL). One ormore communication connections 1650 refers to the hardware/softwareemployed to connect the network interface 1648 to the system bus 1618.While communication connection 1650 is shown for illustrative clarityinside computer 1612, it can also be external to computer 1612. Thehardware/software for connection to the network interface 1648 can alsoinclude, for exemplary purposes only, internal and external technologiessuch as, modems including regular telephone grade modems, cable modemsand DSL modems, ISDN adapters, and Ethernet cards.

Embodiments of the present invention can be a system, a method, anapparatus and/or a computer program product at any possible technicaldetail level of integration. The computer program product can include acomputer readable storage medium (or media) having computer readableprogram instructions thereon for causing a processor to carry outaspects of the present invention. The computer readable storage mediumcan be a tangible device that can retain and store instructions for useby an instruction execution device. The computer readable storage mediumcan be, for example, but is not limited to, an electronic storagedevice, a magnetic storage device, an optical storage device, anelectromagnetic storage device, a semiconductor storage device, or anysuitable combination of the foregoing. A non-exhaustive list of morespecific examples of the computer readable storage medium can alsoinclude the following: a portable computer diskette, a hard disk, arandom access memory (RAM), a read-only memory (ROM), an erasableprogrammable read-only memory (EPROM or Flash memory), a static randomaccess memory (SRAM), a portable compact disc read-only memory (CD-ROM),a digital versatile disk (DVD), a memory stick, a floppy disk, amechanically encoded device such as punch-cards or raised structures ina groove having instructions recorded thereon, and any suitablecombination of the foregoing. A computer readable storage medium, asused herein, is not to be construed as being transitory signals per se,such as radio waves or other freely propagating electromagnetic waves,electromagnetic waves propagating through a waveguide or othertransmission media (e.g., light pulses passing through a fiber-opticcable), or electrical signals transmitted through a wire.

Computer readable program instructions described herein can bedownloaded to respective computing/processing devices from a computerreadable storage medium or to an external computer or external storagedevice via a network, for example, the Internet, a local area network, awide area network and/or a wireless network. The network can includecopper transmission cables, optical transmission fibers, wirelesstransmission, routers, firewalls, switches, gateway computers and/oredge servers. A network adapter card or network interface in eachcomputing/processing device receives computer readable programinstructions from the network and forwards the computer readable programinstructions for storage in a computer readable storage medium withinthe respective computing/processing device. Computer readable programinstructions for carrying out operations of various aspects of thepresent invention can be assembler instructions,instruction-set-architecture (ISA) instructions, machine instructions,machine dependent instructions, microcode, firmware instructions,state-setting data, configuration data for integrated circuitry, oreither source code or object code written in any combination of one ormore programming languages, including an object oriented programminglanguage such as Smalltalk, C++, or the like, and procedural programminglanguages, such as the “C” programming language or similar programminglanguages. The computer readable program instructions can executeentirely on the user's computer, partly on the user's computer, as astand-alone software package, partly on the user's computer and partlyon a remote computer or entirely on the remote computer or server. Inthe latter scenario, the remote computer can be connected to the user'scomputer through any type of network, including a local area network(LAN) or a wide area network (WAN), or the connection can be made to anexternal computer (for example, through the Internet using an InternetService Provider). In some embodiments, electronic circuitry including,for example, programmable logic circuitry, field-programmable gatearrays (FPGA), or programmable logic arrays (PLA) can execute thecomputer readable program instructions by utilizing state information ofthe computer readable program instructions to customize the electroniccircuitry, in order to perform aspects of the present invention.

Aspects of the present invention are described herein with reference toflowchart illustrations and/or block diagrams of methods, apparatus(systems), and computer program products according to embodiments of theinvention. It will be understood that each block of the flowchartillustrations and/or block diagrams, and combinations of blocks in theflowchart illustrations and/or block diagrams, can be implemented bycomputer readable program instructions. These computer readable programinstructions can be provided to a processor of a general purposecomputer, special purpose computer, or other programmable dataprocessing apparatus to produce a machine, such that the instructions,which execute via the processor of the computer or other programmabledata processing apparatus, create means for implementing thefunctions/acts specified in the flowchart and/or block diagram block orblocks. These computer readable program instructions can also be storedin a computer readable storage medium that can direct a computer, aprogrammable data processing apparatus, and/or other devices to functionin a particular manner, such that the computer readable storage mediumhaving instructions stored therein includes an article of manufactureincluding instructions which implement aspects of the function/actspecified in the flowchart and/or block diagram block or blocks. Thecomputer readable program instructions can also be loaded onto acomputer, other programmable data processing apparatus, or other deviceto cause a series of operational acts to be performed on the computer,other programmable apparatus or other device to produce a computerimplemented process, such that the instructions which execute on thecomputer, other programmable apparatus, or other device implement thefunctions/acts specified in the flowchart and/or block diagram block orblocks.

The flowchart and block diagrams in the Figures illustrate thearchitecture, functionality, and operation of possible implementationsof systems, methods, and computer program products according to variousembodiments of the present invention. In this regard, each block in theflowchart or block diagrams can represent a module, segment, or portionof instructions, which includes one or more executable instructions forimplementing the specified logical function(s). In some alternativeimplementations, the functions noted in the blocks can occur out of theorder noted in the Figures. For example, two blocks shown in successioncan, in fact, be executed substantially concurrently, or the blocks cansometimes be executed in the reverse order, depending upon thefunctionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts or carry out combinations of special purpose hardwareand computer instructions.

While the subject matter has been described above in the general contextof computer-executable instructions of a computer program product thatruns on a computer and/or computers, those skilled in the art willrecognize that this disclosure also can or can be implemented incombination with other program modules. Generally, program modulesinclude routines, programs, components, data structures, etc. thatperform particular tasks and/or implement particular abstract datatypes. Moreover, those skilled in the art will appreciate that theinventive computer-implemented methods can be practiced with othercomputer system configurations, including single-processor ormultiprocessor computer systems, mini-computing devices, mainframecomputers, as well as computers, hand-held computing devices (e.g., PDA,phone), microprocessor-based or programmable consumer or industrialelectronics, and the like. The illustrated aspects can also be practicedin distributed computing environments where tasks are performed byremote processing devices that are linked through a communicationsnetwork. However, some, if not all aspects of this disclosure can bepracticed on stand-alone computers. In a distributed computingenvironment, program modules can be located in both local and remotememory storage devices.

As used in this application, the terms “component,” “system,”“platform,” “interface,” and the like, can refer to and/or can include acomputer-related entity or an entity related to an operational machinewith one or more specific functionalities. The entities disclosed hereincan be either hardware, a combination of hardware and software,software, or software in execution. For example, a component can be, butis not limited to being, a process running on a processor, a processor,an object, an executable, a thread of execution, a program, and/or acomputer. By way of illustration, both an application running on aserver and the server can be a component. One or more components canreside within a process and/or thread of execution and a component canbe localized on one computer and/or distributed between two or morecomputers. In another example, respective components can execute fromvarious computer readable media having various data structures storedthereon. The components can communicate via local and/or remoteprocesses such as in accordance with a signal having one or more datapackets (e.g., data from one component interacting with anothercomponent in a local system, distributed system, and/or across a networksuch as the Internet with other systems via the signal). As anotherexample, a component can be an apparatus with specific functionalityprovided by mechanical parts operated by electric or electroniccircuitry, which is operated by a software or firmware applicationexecuted by a processor. In such a case, the processor can be internalor external to the apparatus and can execute at least a part of thesoftware or firmware application. As yet another example, a componentcan be an apparatus that provides specific functionality throughelectronic components without mechanical parts, wherein the electroniccomponents can include a processor or other means to execute software orfirmware that confers at least in part the functionality of theelectronic components. In an aspect, a component can emulate anelectronic component via a virtual machine, e.g., within a cloudcomputing system.

In addition, the term “or” is intended to mean an inclusive “or” ratherthan an exclusive “or.” That is, unless specified otherwise, or clearfrom context, “X employs A or B” is intended to mean any of the naturalinclusive permutations. That is, if X employs A; X employs B; or Xemploys both A and B, then “X employs A or B” is satisfied under any ofthe foregoing instances. Moreover, articles “a” and “an” as used in thesubject specification and annexed drawings should generally be construedto mean “one or more” unless specified otherwise or clear from contextto be directed to a singular form. As used herein, the terms “example”and/or “exemplary” are utilized to mean serving as an example, instance,or illustration. For the avoidance of doubt, the subject matterdisclosed herein is not limited by such examples. In addition, anyaspect or design described herein as an “example” and/or “exemplary” isnot necessarily to be construed as preferred or advantageous over otheraspects or designs, nor is it meant to preclude equivalent exemplarystructures and techniques known to those of ordinary skill in the art.

As it is employed in the subject specification, the term “processor” canrefer to substantially any computing processing unit or deviceincluding, but not limited to, single-core processors; single-processorswith software multithread execution capability; multi-core processors;multi-core processors with software multithread execution capability;multi-core processors with hardware multithread technology; parallelplatforms; and parallel platforms with distributed shared memory.Additionally, a processor can refer to an integrated circuit, anapplication specific integrated circuit (ASIC), a digital signalprocessor (DSP), a field programmable gate array (FPGA), a programmablelogic controller (PLC), a complex programmable logic device (CPLD), adiscrete gate or transistor logic, discrete hardware components, or anycombination thereof designed to perform the functions described herein.Further, processors can exploit nano-scale architectures such as, butnot limited to, molecular and quantum-dot based transistors, switchesand gates, in order to optimize space usage or enhance performance ofuser equipment. A processor can also be implemented as a combination ofcomputing processing units. In this disclosure, terms such as “store,”“storage,” “data store,” data storage,” “database,” and substantiallyany other information storage component relevant to operation andfunctionality of a component are utilized to refer to “memorycomponents,” entities embodied in a “memory,” or components including amemory. It is to be appreciated that memory and/or memory componentsdescribed herein can be either volatile memory or nonvolatile memory, orcan include both volatile and nonvolatile memory. By way ofillustration, and not limitation, nonvolatile memory can include readonly memory (ROM), programmable ROM (PROM), electrically programmableROM (EPROM), electrically erasable ROM (EEPROM), flash memory, ornonvolatile random access memory (RAM) (e.g., ferroelectric RAM (FeRAM).Volatile memory can include RAM, which can act as external cache memory,for example. By way of illustration and not limitation, RAM is availablein many forms such as synchronous RAM (SRAM), dynamic RAM (DRAM),synchronous DRAM (SDRAM), double data rate SDRAM (DDR SDRAM), enhancedSDRAM (ESDRAM), Synchlink DRAM (SLDRAM), direct Rambus RAM (DRRAM),direct Rambus dynamic RAM (DRDRAM), and Rambus dynamic RAM (RDRAM).Additionally, the disclosed memory components of systems orcomputer-implemented methods herein are intended to include, withoutbeing limited to including, these and any other suitable types ofmemory.

What has been described above include mere examples of systems, computerprogram products and computer-implemented methods. It is, of course, notpossible to describe every conceivable combination of components,products and/or computer-implemented methods for purposes of describingthis disclosure, but one of ordinary skill in the art can recognize thatmany further combinations and permutations of this disclosure arepossible. Furthermore, to the extent that the terms “includes,” “has,”“possesses,” and the like are used in the detailed description, claims,appendices and drawings such terms are intended to be inclusive in amanner similar to the term “comprising” as “comprising” is interpretedwhen employed as a transitional word in a claim. The descriptions of thevarious embodiments have been presented for purposes of illustration,but are not intended to be exhaustive or limited to the embodimentsdisclosed.

What is claimed is:
 1. An apparatus, comprising: a housing; and amicrofluidic chip contained within the housing, wherein the microfluidicchip comprises: a separation unit that separates, using one or more nanodeterministic lateral displacement (nanoDLD) arrays, target biologicalentities having a defined size range from other biological entitiesincluded in a biological fluid sample; and a detection unit thatfacilitates detecting presence of one or more biomarkers associated withthe target biological entities using one or more detection molecules ormacromolecules that chemically reacts with the one or more biomarkers.2. The apparatus of claim 1, wherein the target biological entitiescomprise exosomes.
 3. The apparatus of claim 1, wherein the defined sizerange comprises 10.0 nanometers to 200 nanometers.
 4. The apparatus ofclaim 1, wherein the one or more detection molecules or macromoleculescomprise an antibody or aptamer that binds with a target epitope of theone or more biomarkers.
 5. The apparatus of claim 1, wherein thedetection unit comprises a sensing element and wherein a surface of thesensing element is coated with the one or more detection molecules ormacromolecules.
 6. The apparatus of claim 5, wherein the sensing elementcomprises a signal enhancing structure selected from a group consistingof a photonic grating structure, a photonic pillar array structure, anoptoelectrical structure, and a plasmonic structure.
 7. The apparatus ofclaim 5, wherein the one or more detection molecules or macromoleculeschemically react with the one or more biomarkers by binding to the oneor more detection molecules or macromolecules, and wherein based on thebinding, the one or more detection molecules or macromolecules generatea visual signal.
 8. The apparatus of claim 7, wherein a portion of thehousing formed adjacent to the sensing element is transparent orpartially transparent and enables visual observation of the visualsignal.
 9. The apparatus of claim 5, wherein the microfluidic chipfurther comprises: at least one conduit from the separation unit to thedetection unit that facilitates passage of buffer fluid comprising thepurified and isolated target biological entities, as separated from theother biological entities, from the separation unit to the detectionunit; and at least one inlet through which the buffer fluid passes fromthe conduit to the surface of the sensing element.
 10. The apparatus ofclaim 9, wherein the detection unit further comprises a blocking elementformed at an interface between the surface of the sensing element andthe at least one inlet, wherein the blocking element inhibits reverseflow of one or more reacted molecular complexes and exosomes from thesurface of the sensing element through the at least one inlet, whereinthe one or more reacted molecular complexes are formed as a result of achemical reaction between the one or more detection molecules ormacromolecules and the one or more biomarkers.
 11. The apparatus ofclaim 9, wherein the microfluidic chip further comprises: at least oneoutlet from which the buffer fluid and unreacted portions of the targetbiological entities that fail to chemically react with the one or moredetection molecules or macromolecules, are excreted from the detectionunit.
 12. The apparatus of claim 5, wherein the microfluidic chipfurther comprises: at least one inlet via which solution comprising theone or more detection molecules or macromolecules are injected into thedetection unit to coat the surface of the sensing element.
 13. Theapparatus of claim 1, wherein the detection unit comprises two or moreseparate detection chambers, wherein respective chambers of the two ormore separate detection chambers comprise different types of detectionmolecules or macromolecules of the one or more detection molecules ormacromolecules, and wherein the different types of detection moleculesor macromolecules chemically react with different types of biomarkers ofthe one or more biomarkers.
 14. A method comprising: isolating targetbiological entities having a defined size range from other biologicalentities included in a biological fluid sample using a separation unitcomprising one or more nano deterministic lateral displacement (nanoDLD)arrays formed on a microfluidic chip, thereby resulting in isolatedtarget biological entities; driving flow of a buffer fluid comprisingthe isolated target biological entities through a conduit of themicrofluidic chip from the separation unit to a sensing element formedon the microfluidic chip; and facilitating detection of presence of oneor more biomarkers associated with the isolated target biologicalentities based on whether a detectable signal is generated at thesensing element in response to the driving.
 15. The method of claim 14,wherein the isolated target biological entities comprise exosomes. 16.The method of claim 14, wherein the sensing element comprises one ormore detection molecules or macromolecules and wherein the detectablesignal comprises a reaction signal that is indicative of a chemicalinteraction between the one or more detection molecules ormacromolecules and the one or more biomarkers.
 17. The method of claim16, wherein the chemical interaction is selected from a group consistingof: a covalent bonding reaction, an electrostatic interaction, ahydrophobic interaction, an antibody-epitope interaction, anaptamer-epitope reaction, a protein-protein interaction, a protein-smallmolecule interaction, a polymerization reaction, a complementarityreaction, a complementary deoxyribonucleic acid (DNA) strandhybridization interaction, and a complementary ribonucleic acid (RNA)strand hybridization interaction.
 18. The method of claim 16, whereinthe method further comprises: prior to the driving, functionalizing asurface of the sensing element with the one or more detection moleculesor macromolecules, wherein the functionalizing comprises injecting asolution comprising the one or more detection molecules ormacromolecules into a chamber enclosing the surface of sensing elementvia at least one injection inlet of the microfluidic chip.
 19. Themethod of claim 14, wherein the detectable signal comprises a visualsignal and wherein the method further comprises: determining whether thedetectable signal is generated using a microscope positioned adjacentthe sensing element.
 20. The method of claim 14, wherein the detectablesignal comprises a visual signal and wherein the method furthercomprises: capturing, by a device operatively coupled to a processor,image data of the sensing element in association with the driving; anddetermining, by the device, whether the visual signal is generated basedon the image data.
 21. An apparatus, comprising: a housing; and amicrofluidic chip contained within the housing, wherein the microfluidicchip comprises: a separation unit that separates, using one or more nanodeterministic lateral displacement (nanoDLD) arrays, exosomes from otherbiological entities included in a biological fluid sample, resulting inisolated exomes; a detection unit that facilitates detecting presence ofdifferent biomarkers located on or within with the exosomes usingdifferent detection entities that respectively chemically react with thedifferent biomarkers, wherein the different detection entities areselected from a group consisting of molecules and macromolecules; and atleast one channel from the separation unit to the detection unit thatfacilitates flow of a buffer solution comprising the isolated exomes tothe detection unit.
 22. The apparatus of claim 21, wherein the detectionunit comprises different chambers that respectively detect presence of adifferent type of biomarker of the different types of biomarkers, andwherein the different chambers are respectively coated with a differentdetection entity of the different detection entities.
 23. A system,comprising: a microfluidic chip contained within a housing, wherein themicrofluidic chip comprises: a separation unit that separates, using oneor more nano deterministic lateral displacement (nanoDLD) arrays, targetbiological entities having a defined size range from other biologicalentities included in a biological fluid sample, resulting in isolatedtarget biological entities; a detection unit that facilitates detectingpresence of one or more biomarkers associated with the isolated targetbiological entities using one or more detection molecules ormacromolecules that chemically react with the one or more biomarkers;and at least one channel from the separation unit to the detection unitthat facilitates flow of a buffer solution comprising the isolatedtarget biological entities to the detection unit; and an imaging devicethat captures image data in association with flow of the buffer solutionto the detection unit and contact of the buffer solution with the one ormore detection molecules or macromolecules.
 24. The system of claim 23,further comprising: a memory that stores computer executable components;and a processor that executes the computer executable components storedin the memory, wherein the computer executable components comprise: ananalysis component that evaluates the image data to determine biomarkerinformation regarding the presence of the one or more biomarkers; and adiagnosis component that determines diagnostic information regarding amedical condition of a patient from which the biological fluid issampled from based on the biomarker information.
 25. A methodcomprising: isolating target biological entities having a defined sizerange from other biological entities included in a biological fluidsample using a separation unit comprising one or more nano deterministiclateral displacement (nanoDLD) arrays, thereby resulting in isolatedtarget biological entities, wherein the separation unit is formed on amicrofluidic chip contained within a housing; driving flow of a bufferfluid comprising the isolated target biological entities through aconduit of the microfluidic chip from the separation unit to a sensingelement formed on the microfluidic chip, wherein the sensing elementgenerates a visual signal in response to detecting presence of one ormore defined biomarkers associated with the target biological entities;and capturing image data of the detection unit in association with thedriving.